Methods of Identifying Homologous Genes Using FISH

ABSTRACT

The present invention relates to methods of hybridizing nucleic acid probes to genomic DNA.

RELATED APPLICATION DATA

This application is a continuation application which claims priority toU.S. patent application Ser. No. 15/399,788, filed on Jan. 6, 2017,which is a continuation application which claims priority to U.S. patentapplication Ser. No. 14/204,498, filed on Mar. 11, 2014, which claimsthe benefit of Provisional application 61/790,387 and filed Mar. 15,2013 each of which are hereby incorporated by reference in theirentireties.

STATEMENT OF GOVERNMENT INTERESTS

This invention was made with government support under 1 R01GM085169-01A1 awarded by the NIH and 5DP1GM106412-02 awarded by the NIH.The government has certain rights in the invention.

FIELD

The present invention relates in general to the use of oligonucleotideprobes to hybridize to genomic DNA, for example, in a chromosome usingfluorescence in situ hybridization. The methods described herein aredirected to distinguishing homologous genes and chromosomes usingfluorescence in situ hybridization.

BACKGROUND

Fluorescence in situ hybridization (FISH) is a powerful technologywherein nucleic acids are targeted by fluorescently labeled probes andthen visualized via microscopy. FISH is a single-cell assay, making itespecially powerful for the detection of rare events that might beotherwise lost in mixed or asynchronous populations of cells. Inaddition, because FISH is applied to fixed cell or tissue samples, itcan reveal the positioning of chromosomes relative to nuclear,cytoplasmic, and even tissue structures, especially when applied inconjunction with immunofluorescent targeting of cellular components.FISH can also be used to visualize RNA, making it possible forresearchers to simultaneously assess gene expression, chromosomeposition, and protein localization.

Labeled probes in FISH methods bind to a portion of genomic DNA that hasseparated into two strands. The labeled probe binds to one of thestrands. However, distinguishing between homologous genes or chromosomesusing standard FISH methods would be useful. Therefore, methods ofdistinguishing between homologous genes or chromosomes are provided.

SUMMARY

According to standard FISH methods, a portion of double stranded genomicDNA separates and a labeled probe hybridizes to one of the separatedstrands. The labeled probe can then be imaged. Embodiments of thepresent disclosure are directed to methods of distinguishing betweenhomologous nucleic acids, homologous genes or homologous chromosomes ofgenomic DNA using in situ hybridization (“ISH”), such as fluorescence insitu hybridization (“FISH”) as described herein. Embodiments of thepresent disclosure are directed to methods of distinguishing betweenhomologous RNAs or RNAs of different sequence using in situhybridization (“ISH”), such as fluorescence in situ hybridization(“FISH”) as described herein. According to one aspect, the homologousnucleic acids, genes or chromosomes may be maternal nucleic acids, genesor chromosomes and paternal nucleic acids, genes or chromosomes.According to one aspect, a maternal nucleic acid, gene or chromosome isdistinguished from a paternal nucleic acid, gene or chromosome.According to one aspect, a maternal nucleic acid, gene or chromosome isdifferentially labeled from a paternal nucleic acid, gene or chromosome.According to one aspect, the differentially labeled maternal nucleicacid, gene or chromosome and the differentially labeled paternal nucleicacid, gene or chromosome are imaged and are visually distinguishable andidentifiable.

According to one aspect, an ISH or FISH method is provided todistinguish between homologous nucleic acids, genes or chromosomeswhereby differentially labeled sequence differences such as singlenucleotide polymorphisms (“SNP” or “SNPs”) on the maternal nucleic acid,gene or chromosome and differentially labeled sequences differences,such as single nucleotide polymorphisms on the paternal nucleic acid,gene or chromosome are imaged and distinguished. SNPs can be representedby one of A, T, G or C on either the maternal nucleic acid, gene orchromosome or the paternal nucleic acid, gene or chromosome. Accordingto a further aspect, SNPs are identified on both homologous nucleicacids, genes or chromosomes. A first SNP on a first nucleic acid, geneor chromosome, such as a maternal nucleic acid, gene or chromosome isidentified. The nucleotide identifying the SNP is designated the firstnucleotide type and all such SNPs of the first nucleotide type in thefirst nucleic acid, gene or chromosome are hybridized with a firstlabeled nucleotide, thereby identifying the SNP through thecomplementary first labeled nucleotide. It is to be understood that theterm “nucleotide type” refers to the single base designation for aplurality of SNPs. Therefore, the present disclosure contemplateslabeling many SNPs of the same nucleotide type. For example, a pluralityof SNPs are identified by the nucleic acid “A”, thus all of the SNPswould be of the same nucleotide type “A”.

The first labeled nucleotide may be part of an oligonucleotide probeknown to those of skill in the art as being useful with FISH methods.Such probes may also include those referred to as oligopaints andtoe-hold probes. According to one aspect, the term “labeled probe”refers to both a single molecule including a probe sequence and a labelattached thereto, such as by covalent attachment, or a probe sequenceand a separate label component which are added as separate species butthen combine to form a labeled probe. Such an embodiment may be referredto as a secondary label. Wherever reference is made to hybridization ofa labeled nucleotide, such hybridization may be accomplished with thelabeled nucleotide being part of a hybridization probe. SNPs of a secondnucleotide type which is different from the first nucleotide type in thesecond nucleic acid, gene or chromosome, which is a homolog of the firstnucleic acid, gene or chromosome, are hybridized with a second labelednucleotide, thereby identifying the SNP through the complementary secondlabeled nucleotide. According to one aspect, the label of the firstlabeled nucleotide is different from the label of the second labelednucleotide. According to one aspect, the label of the first labelednucleotide is visually distinguishable from the label of the secondlabeled nucleotide. According to one aspect, the label of the firstlabeled nucleotide is spectrally resolvable from the label of the secondlabeled nucleotide. According to one aspect, the first nucleic acid,gene or chromosome is differentially labeled compared to the secondnucleic acid, gene or chromosome. According to one aspect, the firstnucleic acid, gene or chromosome is distinguishable from the secondnucleic acid, gene or chromosome based on differential labeling of thefirst nucleic acid, gene or chromosome from the second nucleic acid,gene or chromosome.

According to an additional aspect, since there are 4 basic naturallyoccurring nucleotides, SNPs in the first nucleic acid, gene orchromosome of a third nucleotide type, which is different from the firstnucleotide type and the second nucleotide type, are hybridized with athird labeled nucleotide, thereby identifying the SNP through thecomplementary third labeled nucleotide. According to one aspect, thethird labeled nucleotide has the same label as the first labelednucleotide and is differentially labeled from the second labelednucleotide. According to this aspect, the first nucleic acid, gene orchromosome is differentially labeled from the second nucleic acid, geneor chromosome. According to this aspect, the labeling of the firstnucleic acid, gene or chromosome is augmented.

According to an additional aspect, SNPs in the second nucleic acid, geneor chromosome of a fourth nucleotide type, which is different from thefirst nucleotide type, the second nucleotide type, and the thirdnucleotide type are hybridized with a fourth labeled nucleotide, therebyidentifying the SNP through the complementary fourth labeled nucleotide.According to one aspect, the fourth labeled nucleotide has the samelabel as the third labeled nucleotide and is differentially labeled fromthe third labeled nucleotide. According to this aspect, the firstnucleic acid, gene or chromosome is differentially labeled from thesecond nucleic acid, gene or chromosome. According to this aspect, thelabeling of the first nucleic acid, gene or chromosome and the secondnucleic acid, gene or chromosome is augmented.

According to certain aspects, the methods described herein are notlimited to using SNPs only to distinguish one homolog from anotherhomolog. Aspects of the present disclosure are directed to the use ofany nucleic acid variation of one homolog from another, as thedifferentially-labeled nucleic acid differences between homologsdifferentiate the homologs. For example two homologs may differ at aposition by a single nucleic acid, i.e., a SNP. But two homologs mayalso differ by a sequence of N nucleotides with N being between about 2and about 10 nucleotides or N is greater than 1 nucleotide, greater than2 nucleotides, greater than 5 nucleotides, greater than 10 nucleotidesand so on. Further, one homolog may lack a sequence the other homologincludes, i.e. have a sequence deletion. Likewise, one homolog mayinclude a sequence that the other homolog lacks, i.e. have sequenceinsertion. Also, one homolog may have a breakpoint where two fragmentsare fused together which the other homolog lacks. Also, one homolog mayinclude one or more or a plurality of modified bases relative to theother homolog with which to distinguish one homolog from another.Accordingly, the methods of the present invention contemplate labeling asequence difference between two homologs, which may be a SNP, aninsertion, a deletion, a breakpoint, modified nucleotides and any othersequence or nucleotide difference known to those of skill in the art.According to this aspect, only one of the nucleotides of the sequencedifference need be labeled to differentiate one homolog from the otherhomolog. For example, according to one aspect, the first nucleotidedifference between the two homologs need only be labeled even though itis the first nucleotide defining an inserted sequence or deletedsequence, as this sufficient to distinguish one homolog from another.

According to one aspect, a fluorescence in situ hybridization method ofdistinguishing a first gene in a maternal chromosome from a second genein a paternal chromosome by sequence differences which distinguish thefirst gene from the second gene and wherein the first gene and thesecond gene are homologs including identifying a first sequencedifference within the first gene, hybridizing a first primer typedirectly upstream of the first sequence difference, extending the firstprimer type across the first sequence difference in the presence of afirst polymerase, first extension nucleotides and a first labeledextension nucleotide complementary to a nucleotide in the first sequencedifference, wherein the first labeled extension nucleotide hybridizes tothe nucleotide in the first sequence difference, identifying a secondsequence difference within the second gene and which is different fromthe first sequence difference, hybridizing a second primer type directlyupstream of the second sequence difference, extending the second primertype across the second sequence difference in the presence of a secondpolymerase, second extension nucleotides and a second labeled extensionnucleotide complementary to a nucleotide in the second sequencedifference wherein the second labeled extension nucleotide hybridizes tothe nucleotide in the second sequence difference, wherein the first geneis differentially labeled from the second gene.

According to one aspect, the method further includes identifying a thirdsequence difference within the first gene and which is different fromthe first sequence difference and the second sequence difference,hybridizing a third primer type directly upstream of the third sequencedifference, extending the third primer type across the third sequencedifference in the presence of a third polymerase, third extensionnucleotides and a third labeled extension nucleotide complementary to anucleotide in the third sequence difference, wherein the third labeledextension nucleotide hybridizes to the nucleotide in the third sequencedifference, and wherein the first gene is differentially labeled fromthe second gene.

According to one aspect, the method includes identifying a thirdsequence difference within the first gene and which is different fromthe first sequence difference and the second sequence difference,hybridizing a third primer type directly upstream of the third sequencedifference, extending the third primer type across the third sequencedifference in the presence of a third polymerase, third extensionnucleotides and a third labeled extension nucleotide complementary to anucleotide in the third sequence difference, wherein the third labeledextension nucleotide hybridizes to the nucleotide in the third sequencedifference, and wherein the first gene is differentially labeled fromthe second gene.

According to one aspect, the method includes identifying a thirdnucleotide type that indicates a third sequence difference within thefirst gene and which is different from the first sequence difference andthe second sequence difference, hybridizing a third primer type directlyupstream of the third sequence difference, extending the third primertype across the third sequence difference in the presence of a thirdpolymerase, third extension nucleotides and a third labeled extensionnucleotide complementary to a nucleotide in the third sequencedifference, wherein the third labeled extension nucleotide hybridizes tothe nucleotide in the third sequence difference, identifying a fourthnucleotide type that indicates a fourth sequence difference within thesecond gene and which is different from the first sequence difference,the second sequence difference, and the third sequence difference,hybridizing a fourth primer type directly upstream of the fourthsequence difference, extending the fourth primer type across the fourthsequence difference in the presence of a fourth polymerase, fourthextension nucleotides and a fourth labeled extension nucleotidecomplementary a nucleotide in the fourth sequence difference wherein thefourth labeled extension nucleotide hybridizes to the nucleotide in thefourth sequence difference, wherein the first gene is differentiallylabeled from the second gene.

According to certain aspects, the term “label” is not limited to adetectable label, but may also include any moiety attached to thenucleotide which can carry out a particular or desired function.Accordingly, a label may be a functional moiety and a nucleotide bearinga functional moiety is referred to as a labeled nucleotide.Detectability, such as by imaging, is just an exemplary function for alabel. According to certain aspects, a labeled nucleotide may bedirectly labeled with a detectable label or indirectly labeled with adetectable label. In either situation, the term “labeled nucleotide”refers to both the directly labeled nucleotide and the nucleotidebearing a moiety capable of attaching a detectable label. In thismanner, the term “label” includes an attachment moiety. Further, thelabeled nucleotide may be a non-naturally occurring nucleotide, i.e. onewhich does not find its source in nature, which may or may not include afunctional moiety. Further, the labeled nucleotide may be a syntheticnucleotide which may or may not include a functional moiety. Further,the labeled nucleotide may be a modified nucleotide which is understoodto be a nucleotide which differs from the standard nucleotides A, C, Gand T by the addition or deletion of a moiety. According to this aspect,the modified nucleotide differs from a standard nucleotide by more thana label. Such modified nucleotides, i.e. methylated bases,hydroxymethylated bases etc., may be naturally occurring ornon-naturally occurring. “Non-naturally occurring” means that the basesdo not find their source in nature.

According to certain aspects, a labeled nucleotide may be hybridized toa SNP by attaching a primer adjacent and upstream of the SNP and thenextending across the SNP using methods known to those of skill in theart with the labeled nucleotide being part of the extending nucleic acidand hybridizing to the SNP. As such, the SNP is then detectably labeledinsofar as the labeled nucleotide identifies the SNP. According to oneaspect, all SNPs of the first nucleic acid, gene or chromosome may belabeled with the same labeled nucleotide and all SNPs of the secondnucleic acid, gene or chromosome may be labeled with the same labelednucleotide. According to one aspect, the labels of the first nucleicacid, gene or chromosome may all be different from the labels of thesecond nucleic acid, gene or chromosome, resulting in differentiallabeling of the first nucleic acid, gene or chromosome and the secondnucleic acid, gene or chromosome and distinguishing of one homologousnucleic acid, gene or chromosome from the other homologous nucleic acid,gene or chromosome. It is to be understood that any combination oflabels may be used to distinguish one homologous nucleic acid, gene orchromosome from the other homologous nucleic acid, gene or chromosomeaccording to the methods described herein. For example, different labelpatterns or spectrally resolvable labels may be used to distinguish onehomologous nucleic acid, gene or chromosome from the other homologousnucleic acid, gene or chromosome. Other methods of using labels todistinguish nucleic acids are known to those of skill in the art. Inaddition, all SNPs of the first nucleic acid, gene or chromosome may belabeled with the same non-naturally occurring nucleotide and all SNPs ofthe second nucleic acid, gene or chromosome may be labeled with the samenon-naturally occurring nucleotide. According to one aspect, thenon-naturally occurring nucleotide of the first nucleic acid, gene orchromosome is different from the non-naturally occurring nucleotide ofthe second nucleic acid, gene or chromosome. According to one aspect, alabeled nucleotide, which may be a non-naturally occurring nucleotidebearing a detectable label, is capable of binding in series with thesame or similar labeled nucleotides thereby creating a chain of labelednucleotides with one or more of the labeled nucleotides bearing adetectable label. In this manner, detection of the SNP may be augmented.According to one aspect, a labeled nucleotide, which may be anon-naturally occurring nucleotide bearing a detectable label, iscapable of binding in series to one or more non-naturally occurringnucleotides hybridized to SNPs on a particular one of the first orsecond nucleic acid, gene or chromosome.

According to one aspect, methods are described herein for distinguishinghomologous nucleic acids, genes or chromosomes based on sequencedifferences. According to this aspect, sequence differences between thefirst nucleic acid, gene or chromosome and the second nucleic acid, geneor chromosome may be differentially labeled and therefore distinguishingthe first nucleic acid, gene or chromosome from the second nucleic acid,gene or chromosome based on the methods described herein.

According to one aspect, a labeled nucleotide may be hybridized to a SNPby ligating two oligonucleotides across the SNP using methods known tothose of skill in the art with the labeled nucleotide being part of oneof the two oligonucleotides and hybridizing to the SNP. According tothis aspect, the labeled nucleotide may be a non-naturally occurringnucleotide as described herein.

According to one aspect, SNPs may be amplified using methods known tothose of skill in the art and the resulting amplicons being subjected tothe labeling methods described herein to generate an enhanced detectablesignal. The amplicons are representative of the SNPs present on eitherthe first or second nucleic acid, gene or chromosome. Accordingly, theiridentification using the methods described herein is used to distinguishhomologous nucleic acids, gene or chromosomes.

According to one aspect, certain nucleic acid probes may be labeled orunlabeled. Certain nucleic acid probes may be directly labeled orindirectly labeled. According to certain aspects, nucleic acid probesmay include a primary nucleic acid sequence that is non-hybridizable toa target nucleic acid sequence. According to certain aspects, theprimary nucleic acid sequence is hybridizable with a secondary nucleicacid sequence. According to certain aspects, the secondary nucleic acidsequence may include a label. According to this aspect, the nucleic acidprobes are indirectly labeled as the secondary nucleic acid binds to theprimary nucleic acid thereby indirectly labeling the probe whichhybridizes to the target nucleic acid sequence. According to certainaspects, the secondary nucleic acid sequence hybridizes with the primarynucleic acid sequence to create a recognition sequence which may berecognized or bound by a functional moiety. According to certainaspects, a plurality of nucleic acid probes are provided with eachhaving a common primary nucleic acid sequence. That is, the primarynucleic acid sequence is common to a plurality of nucleic acid probes,such that each nucleic acid probe in the plurality has the same orsubstantially similar primary nucleic acid sequence. In this manner, aplurality of common secondary nucleic acid sequences are provided whichhybridize to the plurality of common primary nucleic acid sequences.That is, each secondary nucleic acid sequence has the same orsubstantially similar nucleic acid sequence. According to one exemplaryembodiment, a single primary nucleic acid sequence is provided for eachof the nucleic acid probes in the plurality. Accordingly, only a singlesecondary nucleic acid sequence which is hybridizable to the primarynucleic acid sequence need be provided to label each of the nucleic acidprobes. According to certain aspects, the common secondary nucleic acidsequences may include a common label. According to this aspect, aplurality of nucleic acid probes are provided having substantiallydiverse nucleic acid sequences hybridizable to different target nucleicacid sequences and where the plurality of nucleic acid probes havecommon primary nucleic acid sequences. Accordingly, a common secondarynucleic acid sequence having a label may be used to indirectly labeleach of the plurality of nucleic acid probes. According to this aspect,a single or common primary nucleic acid sequence and secondary nucleicacid sequence pair can be used to indirectly label diverse nucleic acidprobe sequences. Methods using nucleic acid probes as described hereininclude any method where probe hybridization is useful, including butnot limited to fluorescence in situ hybridization methods known to thoseof skill in the art or any other method where a label, such as afunctional moiety, is desired to be brought to or near a target nucleicacid sequence through hybridization of the probe to the target nucleicacid sequence for detection, chemical modification, retrieving orbinding to a target molecule, or providing other functions.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other features and advantages of the present inventionwill be more fully understood from the following detailed description ofillustrative embodiments taken in conjunction with the accompanyingdrawing in which:

FIG. 1 is a schematic representation of a maternal homolog and apaternal homolog.

FIG. 2 is a schematic representation of a first SNP having a firstnucleotide type A on the maternal homolog.

FIG. 3 is a schematic representation of a second SNP having a secondnucleotide type G being different from the first nucleotide type andbeing on the paternal homolog.

FIG. 4 is a schematic representation of third SNP having a thirdnucleotide type T being different from the first nucleotide type and thesecond nucleotide type and being on the maternal homolog and a fourthSNP having a fourth nucleotide type C being different from the firstnucleotide type, the second nucleotide type and the third nucleotidetype and being on the paternal homolog.

FIG. 5 is a schematic representation of a nucleotide A from among thenucleotide type A on the maternal homolog for a SNP being excluded fromlabeling because its counterpart on the paternal homolog is a nucleotidetype for a SNP on the maternal homolog. The same is shown for aparticular G on the paternal homolog.

FIG. 6 is a schematic representation of the usable nucleotides takinginto consideration the exclusion of SNPs shown in FIG. 5.

FIG. 7 is a schematic showing hybridization of probes upstream of SNPsto be used to differentiate the homologs.

FIG. 8 is a schematic showing extension of the probes to cover the SNPswith a labeled nucleotide being complementary to a SNP. The label forthe maternal homolog is shown as being different from the label for thepaternal homolog.

FIG. 9 is a schematic showing extension of the probes to cover the SNPswith a non-naturally occurring nucleotide being complementary to a SNP.The non-naturally occurring nucleotide is shown as being distinct foreach SNP.

FIG. 10 is a schematic showing extension of the probes to cover the SNPswith a non-naturally occurring nucleotide being complementary to a SNP.The non-naturally occurring nucleotides corresponding to the maternalhomolog share a feature “m” which is distinct for the maternal homolog.The non-naturally occurring nucleotides corresponding to the paternalhomolog share a feature “p” which is distinct for the paternal homolog.

FIG. 11 is a schematic showing extension of the probes to cover the SNPswith a non-naturally occurring nucleotide being complementary to a SNP.The non-naturally occurring nucleotides corresponding to the maternalhomolog share a feature “m” which is distinct for the maternal homolog.The non-naturally occurring nucleotides corresponding to the paternalhomolog share a feature “p” which is distinct for the paternal homolog.A detectable label can be added to each non-naturally occurringnucleotide. A first common label for the maternal homolog is shown. Asecond common label for the paternal homolog is shown.

FIG. 12 is a schematic showing extension of the probes to cover the SNPswith a non-naturally occurring nucleotide being complementary to a SNP.The non-naturally occurring nucleotides corresponding to the maternalhomolog share a feature “m” which is distinct for the maternal homolog.The non-naturally occurring nucleotides corresponding to the paternalhomolog share a feature “p” which is distinct for the paternal homolog.A detectable label can be added to each non-naturally occurringnucleotide. Each non-naturally occurring nucleotide can be extended witha non-naturally occurring nucleotide, for example, in series, and so asto augment a signal from a SNP. A first common label for the maternalhomolog is shown. A second common label for the paternal homolog isshown.

FIG. 13 is a schematic embodiment of FIG. 12 showing non-naturallyoccurring nucleotides extending from a non-naturally occurringnucleotides corresponding to SNPs on the maternal homolog.

FIG. 14 is a schematic embodiment of FIG. 12 showing a singlenon-naturally occurring nucleotide type extending from two differentnon-naturally occurring nucleotides corresponding to SNPs on thematernal homolog. In this manner, a labeled non-naturally occurringnucleotide can be a dual purpose label.

FIG. 15 is a schematic embodiment of FIG. 12 showing a singlenon-naturally occurring nucleotide type extending from two differentnon-naturally occurring nucleotides corresponding to SNPs on thematernal homolog and showing a single non-naturally occurring nucleotidetype extending from two different non-naturally occurring nucleotidescorresponding to SNPs on the paternal homolog. In this manner, a labelednon-naturally occurring nucleotide can be a dual purpose label.

FIG. 16 is a schematic showing differentiation of a maternal homologfrom a paternal homolog using labeling of sequences which differ betweenthe maternal homolog and the paternal homolog.

FIG. 17 is a schematic showing ligation across SNPs on the maternalhomolog and the paternal homolog with the result being a labelednucleotide being hybridized to a SNP and in a manner to differentiatethe maternal homolog from the paternal homolog.

FIG. 18 is a schematic showing ligation across SNPs on the maternalhomolog and the paternal homolog with the result being a non-naturallyoccurring base being hybridized to a SNP and in a manner todifferentiate the maternal homolog from the paternal homolog.

FIG. 19 is a schematic showing hybridization of a labeled probe to a SNPand in a manner to differentiate the maternal homolog from the paternalhomolog.

FIG. 20 is a schematic showing hybridization of a labeled toe-hold probe(in schematic) to a SNP and in a manner to differentiate the maternalhomolog from the paternal homolog.

FIG. 21 is a schematic showing amplification of a nucleotidecomplementary to a SNP using a padlock probe including the nucleotideand rolling circle amplification.

FIG. 22 is a schematic showing amplification of a nucleotidecomplementary to a SNP using a padlock probe and ligation to include thenucleotide complementary to the SNP into a template for rolling circleamplification.

FIG. 23 is a schematic showing use of a padlock probe to hybridize acomplementary non-naturally occurring nucleotide to a SNP where thepadlock probe hybridizes flanking the SNP. The probe is ligated acrossthe SNP with the inclusion of the complementary non-naturally occurringnucleotide. The non-naturally occurring nucleotide can then be imaged orotherwise detected using the methods described herein and in a manner todistinguish the maternal homolog from the paternal homolog.

DETAILED DESCRIPTION

The practice of certain embodiments or features of certain embodimentsmay employ, unless otherwise indicated, conventional techniques ofmolecular biology, microbiology, recombinant DNA, and so forth which arewithin ordinary skill in the art. Such techniques are explained fully inthe literature. See e.g., Sambrook, Fritsch, and Maniatis, MOLECULARCLONING: A LABORATORY MANUAL, Second Edition (1989), OLIGONUCLEOTIDESYNTHESIS (M. J. Gait Ed., 1984), ANIMAL CELL CULTURE (R. I. Freshney,Ed., 1987), the series METHODS IN ENZYMOLOGY (Academic Press, Inc.);GENE TRANSFER VECTORS FOR MAMMALIAN CELLS (J. M. Miller and M. P. Caloseds. 1987), HANDBOOK OF EXPERIMENTAL IMMUNOLOGY, (D. M. Weir and C. C.Blackwell, Eds.), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F. M. Ausubel,R. Brent, R. E. Kingston, D. D. Moore, J. G. Siedman, J. A. Smith, andK. Struhl, eds., 1987), CURRENT PROTOCOLS IN IMMUNOLOGY (J. E. coligan,A. M. Kruisbeek, D. H. Margulies, E. M. Shevach and W. Strober, eds.,1991); ANNUAL REVIEW OF IMMUNOLOGY; as well as monographs in journalssuch as ADVANCES IN IMMUNOLOGY. All patents, patent applications, andpublications mentioned herein, both supra and infra, are herebyincorporated herein by reference.

Terms and symbols of nucleic acid chemistry, biochemistry, genetics, andmolecular biology used herein follow those of standard treatises andtexts in the field, e.g., Komberg and Baker, DNA Replication, SecondEdition (W.H. Freeman, New York, 1992); Lehninger, Biochemistry, SecondEdition (Worth Publishers, New York, 1975); Strachan and Read, HumanMolecular Genetics, Second Edition (Wiley-Liss, New York, 1999);Eckstein, editor, Oligonucleotides and Analogs: A Practical Approach(Oxford University Press, New York, 1991); Gait, editor, OligonucleotideSynthesis: A Practical Approach (IRL Press, Oxford, 1984); and the like.

It is to be understood that methods steps described herein need not beperformed in the order listed unless expressly stated. Method steps maybe performed in any order. Further, method steps may be performedsimultaneously or together and need not be performed separately orindividually. To the extent that methods describe multiple probes beinghybridized to various nucleic acids on separate homologs, suchhybridization may be performed as a single step with all reagentscombined. Individual hybridization steps need not be performedindividually.

According to embodiments of the present disclosure, a method ofdistinguishing homologs is provided using fluorescence in situhybridization or any methods known to those of skill in the art wherenucleic acid probes are used to hybridize to double stranded DNA where aportion of the double stranded DNA has separated into two separatestrands, i.e. a first strand and a complementary strand, such as ingenomic DNA within a cell or tissue. It is to be understood thatreference to a first strand and a complementary strand is relative whenseparating double stranded nucleic acids. That is, either strand can bethe first strand or the complementary strand.

Selecting one strand as the first strand makes the remaining strand thecomplementary strand.

Exemplary method where homologs can be distinguished utilizefluorescence in situ hybridization or FISH which is a cytogenetictechnique that is used to detect and localize the presence or absence ofspecific DNA sequences on chromosomes. FISH uses fluorescent probes thatbind to only those parts of the chromosome with which they show a highdegree of sequence complementarity. Fluorescence microscopy can be usedto find out where the fluorescent probe is bound to the chromosomes.

Exemplary FISH methods include standard in situ hybridization (ISH)techniques (see, e.g., Gall and Pardue (1981) Meth. Enzymol. 21:470;Henderson (1982) Int. Review of Cytology 76:1). Generally, ISH comprisesthe following major steps: (1) fixation of the biological structure tobe analyzed (e.g., a chromosome spread), (2) pre-hybridization treatmentof the biological structure to increase accessibility of target DNA(e.g., denaturation with heat or alkali), (3) optional pre-hybridizationtreatment to reduce nonspecific binding (e.g., by blocking thehybridization capacity of repetitive sequences), (4) hybridization ofthe mixture of nucleic acids to the nucleic acid in the biologicalstructure or tissue; (5) post-hybridization washes to remove nucleicacid fragments not bound in the hybridization and (6) detection of thehybridized labelled oligonucleotides. The reagents used in each of thesesteps and their conditions of use vary depending on the particularsituation and whether their use is required with any particular probes.Hybridization conditions are also described in U.S. Pat. No. 5,447,841.It will be appreciated that numerous variations of in situ hybridizationprotocols and conditions are known and may be used in conjunction withthe present invention by practitioners following the guidance providedherein.

As used herein, the term “chromosome” refers to the support for thegenes carrying heredity in a living cell, including DNA, protein, RNAand other associated factors. There exists a conventional internationalsystem for identifying and numbering the chromosomes of the humangenome. The size of an individual chromosome may vary within amulti-chromosomal genome and from one genome to another. A chromosomecan be obtained from any species. A chromosome can be obtained from anadult subject, a juvenile subject, an infant subject, from an unbornsubject (e.g., from a fetus, e.g., via prenatal test such asamniocentesis, chorionic villus sampling, and the like or directly fromthe fetus, e.g., during a fetal surgery) from a biological sample (e.g.,a biological tissue, fluid or cells (e.g., sputum, blood, blood cells,tissue or fine needle biopsy samples, urine, cerebrospinal fluid,peritoneal fluid, and pleural fluid, or cells therefrom) or from a cellculture sample (e.g., primary cells, immortalized cells, partiallyimmortalized cells or the like). In certain exemplary embodiments, oneor more chromosomes can be obtained from one or more genera including,but not limited to, Homo, Drosophila, Caenorhabiditis, Danio, Cyprinus,Equus, Canis, Ovis, Ocorynchus, Salmo, Bos, Sus, Gallus, Solanum,Triticum, Oryza, Zea, Hordeum, Musa, Avena, Populus, Brassica, Saccharumand the like.

As used herein, the terms “complementary” and “complementarity” are usedin reference to nucleotide sequences related by the base-pairing rules.For example, the sequence 5′-AGT-3′ is complementary to the sequence5′-ACT-3′. Complementarity can be partial or total. Partialcomplementarity occurs when one or more nucleic acid bases is notmatched according to the base pairing rules. Total or completecomplementarity between nucleic acids occurs when each and every nucleicacid base is matched with another base under the base pairing rules. Thedegree of complementarity between nucleic acid strands has significanteffects on the efficiency and strength of hybridization between nucleicacid strands.

The term “homology” when used in relation to nucleic acids refers to adegree of complementarity. There may be partial homology (i.e., partialidentity) or complete homology (i.e., complete identity). A partiallycomplementary sequence is one that at least partially inhibits acompletely complementary sequence from hybridizing to a target nucleicacid and is referred to using the functional term “substantiallyhomologous.” The inhibition of hybridization of the completelycomplementary sequence to the target sequence may be examined using ahybridization assay (Southern or Northern blot, solution hybridizationand the like) under conditions of low stringency. A substantiallyhomologous sequence or probe (i.e., an oligonucleotide which is capableof hybridizing to another oligonucleotide of interest) will compete forand inhibit the binding (i.e., the hybridization) of a completelyhomologous sequence to a target under conditions of low stringency. Thisis not to say that conditions of low stringency are such thatnon-specific binding is permitted; low stringency conditions requirethat the binding of two sequences to one another be a specific (i.e.,selective) interaction. The absence of non-specific binding may betested by the use of a second target which lacks even a partial degreeof complementarity (e.g., less than about 30% identity); in the absenceof non-specific binding the probe will not hybridize to the secondnon-complementary target.

When used in reference to a double-stranded nucleic acid sequence suchas a cDNA or genomic clone, the term “substantially homologous” refersto any probe which can hybridize to either or both strands of thedouble-stranded nucleic acid sequence under conditions of lowstringency. When used in reference to a single-stranded nucleic acidsequence, the term “substantially homologous” refers to any probe whichcan hybridize to the single-stranded nucleic acid sequence underconditions of low stringency.

Probes included within the scope of the present disclosure include thoseknown to be useful with FISH methods. FISH probes are typically derivedfrom genomic inserts subcloned into vectors such as plasmids, cosmids,and bacterial artificial chromosomes (BACs), or from flow-sortedchromosomes. These inserts and chromosomes can be used to produce probeslabeled directly via nick translation or PCR in the presence offluorophore-conjugated nucleotides or probes labeled indirectly withnucleotide-conjugated haptens, such as biotin and digoxigenin, which canbe visualized with secondary detection reagents. Probe DNA is oftenfragmented into about 150-250 bp pieces to facilitate its penetrationinto fixed cells and tissues. As many genomic clones contain highlyrepetitive sequences, such as SINE and Alu elements, hybridization oftenneeds to be performed in the presence of unlabeled repetitive DNA toprevent off-target hybridizations that increase background signal. Suchprobes may be referred to as “chromosome paints” which refers todetectably labeled polynucleotides that have sequences complementary toDNA sequences from a particular chromosome or sub-chromosomal region ofa particular chromosome. Chromosome paints that are commerciallyavailable are derived from fluorescence activated cell sorted (FACS)and/or flow sorted chromosomes or from bacterial artificial chromosomes(BACs) or yeast artificial chromosomes (YACs).

Many types of custom-synthesized oligonucleotides (oligos) have alsobeen used as FISH probes, including DNA, peptide nucleic acid (PNA), andlocked nucleic acid (LNA) oligos. One advantage of oligo probes is thatthey are designed to target a precisely defined sequence rather thanrelying on the isolation of a clone that is specific for the desiredgenomic target. Also, as these probes are typically short (about 20-50bp) and single-stranded by nature, they efficiently diffuse into fixedcells and tissues and are unhindered by competitive hybridizationbetween complimentary probe fragments. Recently developed methodsutilizing oligo probes have allowed the visualization of single-copyviral DNA as well as individual mRNA molecules using branched DNA signalamplification or a few dozen short oligo probes and, by targetingcontiguous blocks of highly repetitive sequences as a strategy toamplify signal, enabled the first FISH-based genome-wide RNAi screen.Oligo FISH probes have also been generated directly from genomic DNAusing many parallel PCR reactions.

The availability of complex oligo libraries produced by massivelyparallel synthesis has enabled a new generation of oligo-basedtechnologies. These libraries are synthesized on a solid substrate, thenamplified or chemically cleaved in order to move the library intosolution. Popular applications of oligo libraries include targetedcapture for next generation sequencing and custom gene synthesis. Twovery recent studies have used complex libraries to visualize single-copyregions of mammalian genomes by FISH. One study used long oligos (>150bp) as templates for PCR, and then labeled the amplification productsnon-specifically, while the other adapted a 75-100 bp single-strandedsequence-capture library for FISH by replacing the 5′ biotin with afluorophore.

Additional labeled probes include those known as “oligopaints” asdescribed in US 2010/0304994. As used herein, the term “Oligopaint”refers to detectably labeled polynucleotides that have sequencescomplementary to an oligonucleotide sequence, e.g., a portion of a DNAsequence e.g., a particular chromosome or sub-chromosomal region of aparticular chromosome. Oligopaints are generated from synthetic probesand arrays that are, optionally, computationally patterned (rather thanusing natural DNA sequences and/or chromosomes as a template). SinceOligopaints are generated using nucleic acid sequences that are presentin a pool, they are no longer spatially addressable (i.e., no longerattached to an array). Surprisingly, however, this method increasesresolution of the oligopaints over chromosome paints that are made usingyeast artificial chromosomes (YACs), bacterial artificial chromosomes(BACs), and/or flow sorted chromosomes.

In certain exemplary embodiments, small Oligopaints are provided. Asused herein, the term “small Oligopaint” refers to an Oligopaint ofbetween about 5 bases and about 100 bases long, or an Oligopaint ofabout 5 bases, about 10 bases, about 15 bases, about 20 bases, about 25bases, about 30 bases, about 35 bases, about 40 bases, about 45 bases,about 50 bases, about 55 bases, about 60 bases, about 65 bases, about 70bases, about 75 bases, about 80 bases, about 85 bases, about 90 bases,about 95 bases, or about 100 bases. Small Oligopaints can access targetsthat are not accessible to longer oligonucleotide probes. For example,in certain aspects small Oligopaints can pass into a cell, can pass intoa nucleus, and/or can hybridize with targets that are partially bound byone or more proteins, etc. Small Oligopaints are also useful forreducing background, as they can be more easily washed away than largerhybridized oligonucleotide sequences. As used herein, the terms“Oligopainted” and “Oligopainted region” refer to a target nucleotidesequence (e.g., a chromosome) or region of a target nucleotide sequence(e.g., a sub-chromosomal region), respectively, that has hybridizedthereto one or more Oligopaints. Oligopaints can be used to label atarget nucleotide sequence, e.g., chromosomes and sub-chromosomalregions of chromosomes during various phases of the cell cycleincluding, but not limited to, interphase, preprophase, prophase,prometaphase, metaphase, anaphase, telophase and cytokenesis.

According to certain aspects, labeled toe-hold probes are useful in themethods described herein. Toe-hold probes are known to those of skill inthe art as described in Zhang et al., Optimizing the Specificity ofNucleic Acid Hybridization, Nature Chemistry, DOI: 10.1038/NCHEM.1246(published online Jan. 22, 2012) hereby incorporated by reference in itsentirety for all purposes. Such probes are capable of distinguishingbetween sequences which differ by only one nucleotide in a highlyspecific manner. According to one aspect, a toe-hold probe includes aprobe strand and a complementary protector strand hybridized thereto.The probe strand includes a 5′ overhang sequence which is complementaryto the target sequence. The probe strand also includes a 3′ sequencethat is non-hybridizable with the target sequence. [The 3′sequence thatis non-hybridizable with the target sequence is similar in length, basecomposition and thermodynamic binding strength to the 5′ overhangsequence. The probe strand may include a label at the 3′ end or a sitefor attachment of a label, such as in secondary labeling or indirectlabeling. If the probe strand includes a label at the 3′ end, thecomplementary protector strand may include a quencher at the 5′ end inproximity to quench the label, i.e., the label is in a non-active state.The hybridization of the probe to the target sequence initiates throughbinding of the 5′ overhang sequence to the target and proceeds throughbranch migration to hybridize with the remaining portion of the targetsequence and to displace the complementary protector strand. The 3′sequence of the complementary protector strand then spontaneouslydissociates from the probe strand leaving a 3′ overhang sequence. If the3′ sequence includes a site for attachment of a label, then a label canbe added and attached to the probe strand. If the 3′ sequence includes alabel, then the quencher dissociates from the label and the labelbecomes activated.

Nucleic Acid

The terms “nucleic acid,” “nucleic acid molecule,” “nucleic acidsequence,” “nucleic acid fragment,” “oligonucleotide” and“polynucleotide” are used interchangeably and are intended to include,but not limited to, a polymeric form of nucleotides that may havevarious lengths, either deoxyribonucleotides or ribonucleotides, oranalogs thereof. The labeled probes described herein may include or be a“nucleic acid,” “nucleic acid molecule,” “nucleic acid sequence,”“nucleic acid fragment,” “oligonucleotide” or “polynucleotide.”Oligonucleotides or polynucleotides useful in the methods describedherein may comprise natural nucleic acid sequences and variants thereof,artificial nucleic acid sequences, or a combination of such sequences.Oligonucleotides or polynucleotides may be single stranded or doublestranded.

A polynucleotide is typically composed of a specific sequence of fournucleotide bases: adenine (A); cytosine (C); guanine (G); and thymine(T) (uracil (U) for thymine (T) when the polynucleotide is RNA). Thus,the term “polynucleotide sequence” is the alphabetical representation ofa polynucleotide molecule; alternatively, the term may be applied to thepolynucleotide molecule itself. This alphabetical representation can beinput into databases in a computer having a central processing unit andused for bioinformatics applications such as functional genomics andhomology searching. Polynucleotides may optionally include one or morenon-standard nucleotide(s), nucleotide analog(s) and/or modifiednucleotides.

Modified Nucleotides

The terms “nucleotide analog,” “altered nucleotide” and “modifiednucleotide” refer to a non-standard nucleotide, including naturallyoccurring and non-naturally occurring ribonucleotides ordeoxyribonucleotides. In certain exemplary embodiments, nucleotideanalogs are modified at any position so as to alter certain chemicalproperties of the nucleotide yet retain the ability of the nucleotideanalog to perform its intended function. Examples of positions of thenucleotide which may be derivitized include the 5 position, e.g.,5-(2-amino)propyl uridine, 5-bromo uridine, 5-propyne uridine,5-propenyl uridine, etc.; the 6 position, e.g., 6-(2-amino) propyluridine; the 8-position for adenosine and/or guanosines, e.g., 8-bromoguanosine, 8-chloro guanosine, 8-fluoroguanosine, etc. Nucleotideanalogs also include deaza nucleotides, e.g., 7-deaza-adenosine; 0- andN-modified (e.g., alkylated, e.g., N6-methyl adenosine, or as otherwiseknown in the art) nucleotides; and other heterocyclically modifiednucleotide analogs such as those described in Herdewijn, AntisenseNucleic Acid Drug Dev., 2000 Aug. 10(4):297-310.

Nucleotide analogs may also comprise modifications to the sugar portionof the nucleotides. For example the 2′ OH-group may be replaced by agroup selected from H, OR, R, F, Cl, Br, I, SH, SR, NH₂, NHR, NR₂, COOR,or OR, wherein R is substituted or unsubstituted C₁-C₆ alkyl, alkenyl,alkynyl, aryl, etc. Other possible modifications include those describedin U.S. Pat. Nos. 5,858,988, and 6,291,438.

The phosphate group of the nucleotide may also be modified, e.g., bysubstituting one or more of the oxygens of the phosphate group withsulfur (e.g., phosphorothioates), or by making other substitutions whichallow the nucleotide to perform its intended function such as describedin, for example, Eckstein, Antisense Nucleic Acid Drug Dev. 2000 Apr.10(2):117-21, Rusckowski et al. Antisense Nucleic Acid Drug Dev. 2000Oct. 10(5):333-45, Stein, Antisense Nucleic Acid Drug Dev. 2001 Oct.11(5): 317-25, Vorobjev et al. Antisense Nucleic Acid Drug Dev. 2001Apr. 11(2):77-85, and U.S. Pat. No. 5,684,143. Certain of theabove-referenced modifications (e.g., phosphate group modifications)decrease the rate of hydrolysis of, for example, polynucleotidescomprising said analogs in vivo or in vitro.

Examples of modified nucleotides include, but are not limited todiaminopurine, S²T, 5-fluorouracil, 5-bromouracil, 5-chlorouracil,5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine,5-(carboxyhydroxylmethyl)uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-D46-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,2,6-diaminopurine and the like. Nucleic acid molecules may also bemodified at the base moiety (e.g., at one or more atoms that typicallyare available to form a hydrogen bond with a complementary nucleotideand/or at one or more atoms that are not typically capable of forming ahydrogen bond with a complementary nucleotide), sugar moiety orphosphate backbone. Nucleic acid molecules may also containamine-modified groups, such as aminoallyl-dUTP (aa-dUTP) andaminohexhylacrylamide-dCTP (aha-dCTP) to allow covalent attachment ofamine reactive moieties, such as N-hydroxy succinimide esters (NHS).

Non-naturally occurring nucleotides and polymerases which can be usedwith such bases in include those described in Gommers-Ampt et al., TheFASEB Journal, Vol. 9, pp. 1034-1042 (1995); Leconte, et al., J. Am.Chem. Soc; 127(36), pp. 12470-12471 (2005); Leconte et al., Angew. Chem.Int. Ed. 2010, 49, pp. 5921-5924; Malyshev et al., J. Am. Chem. Soc.2009, 131, 14620-14621; Metzker, Genome Research 15:1767-1776 (2005);Metzker, Nature Reviews/Genetics, Vol. 11, pp. 31-46 (2010); and Yang etal., Angew. Chem. Int. Ed, 2010, 49, 177-180 each of which is herebyincorporated by reference in its entirety for all purposes.

In certain exemplary embodiments, nucleotide analogs or derivatives willbe used, such as nucleosides or nucleotides having protecting groups oneither the base portion or sugar portion of the molecule, or havingattached or incorporated labels, or isosteric replacements which resultin monomers that behave in either a synthetic or physiologicalenvironment in a manner similar to the parent monomer. The nucleotidescan have a protecting group which is linked to, and masks, a reactivegroup on the nucleotide. A variety of protecting groups are useful inthe invention and can be selected.

Oligonucleotide sequences, such as single stranded oligonucleotidesequences to be used for labeled probes, may be isolated from naturalsources, synthesized or purchased from commercial sources. In certainexemplary embodiments, oligonucleotide sequences may be prepared usingone or more of the phosphoramidite linkers and/or sequencing by ligationmethods known to those of skill in the art. Oligonucleotide sequencesmay also be prepared by any suitable method, e.g., standardphosphoramidite methods such as those described herein below as well asthose described by Beaucage and Carruthers ((1981) Tetrahedron Lett. 22:1859) or the triester method according to Matteucci et al. (1981) J. Am.Chem. Soc. 103:3185), or by other chemical methods using either acommercial automated oligonucleotide synthesizer or high-throughput,high-density array methods known in the art (see U.S. Pat. Nos.5,602,244, 5,574,146, 5,554,744, 5,428,148, 5,264,566, 5,141,813,5,959,463, 4,861,571 and 4,659,774, incorporated herein by reference inits entirety for all purposes). Pre-synthesized oligonucleotides mayalso be obtained commercially from a variety of vendors.

In certain exemplary embodiments, oligonucleotide sequences may beprepared using a variety of microarray technologies known in the art.Pre-synthesized oligonucleotide and/or polynucleotide sequences may beattached to a support or synthesized in situ using light-directedmethods, flow channel and spotting methods, inkjet methods, pin-basedmethods and bead-based methods set forth in the following references:McGall et al. (1996) Proc. Natl. Acad. Sci. U.S.A. 93:13555; SyntheticDNA Arrays In Genetic Engineering, Vol. 20:111, Plenum Press (1998);Duggan et al. (1999) Nat. Genet. S21:10; Microarrays: Making Them andUsing Them In Microarray Bioinformatics, Cambridge University Press,2003; U.S. Patent Application Publication Nos. 2003/0068633 and2002/0081582; U.S. Pat. Nos. 6,833,450, 6,830,890, 6,824,866, 6,800,439,6,375,903 and 5,700,637; and PCT Application Nos. WO 04/031399, WO04/031351, WO 04/029586, WO 03/100012, WO 03/066212, WO 03/065038, WO03/064699, WO 03/064027, WO 03/064026, WO 03/046223, WO 03/040410 and WO02/24597.

Polymerase recognition sites, cleavage sites and/or label or detectablemoiety addition sites may be added to the single strandedoligonucleotides during synthesis using known materials and methods.

Oligonucleotide Probes

Oligonucleotide probes useful for labeled probes or primers according tothe present disclosure may have any desired nucleotide length andnucleic acid sequence. Accordingly, aspects of the present disclosureare directed to the use of a plurality or set of nucleic acid probes,such as single stranded nucleic acid probes, such as oligonucleotidepaints. Additional labeled probes include those known as “oligopaints”as described in US 2010/0304994. The term “probe” refers to asingle-stranded oligonucleotide sequence that will recognize and form ahydrogen-bonded duplex with a complementary sequence in a target nucleicacid sequence or its cDNA derivative. The probe includes a targethybridizing nucleic acid sequence. Exemplary nucleic acid sequences maybe short nucleic acids or long nucleic acids. Exemplary nucleic acidsequences include oligonucleotide paints. Exemplary nucleic acidsequences are those having between about 1 nucleotide to about 100,000nucleotides, between about 3 nucleotides to about 50,000 nucleotides,between about 5 nucleotides to about 10,000 nucleotides, between about10 nucleotides to about 10,000 nucleotides, between about 10 nucleotidesto about 1,000 nucleotides, between about 10 nucleotides to about 500nucleotide, between about 10 nucleotides to about 100 nucleotides,between about 10 nucleotides to about 70 nucleotides, between about 15nucleotides to about 50 nucleotides, between about 20 nucleotides toabout 60 nucleotides, between about 50 nucleotides to about 500nucleotides, between about 70 nucleotides to about 300 nucleotides,between about 100 nucleotides to about 200 nucleotides, and all rangesor values in between whether overlapping or not. Exemplaryoligonucleotide probes include between about 10 nucleotides to about 100nucleotides, between about 10 nucleotides to about 70 nucleotides,between about 15 nucleotides to about 50 nucleotides, between about 20nucleotides to about 60 nucleotides and all ranges and values in betweenwhether overlapping or not. According to one aspect, oligonucleotideprobes according to the present disclosure should be capable ofhybridizing to a target nucleic acid. Probes according to the presentdisclosure may include a label or detectable moiety as described herein.Oligonucleotides or polynucleotides may be designed, if desired, withthe aid of a computer program such as, for example, DNAWorks, orGene2Oligo.

Oligonucleotide probes according to the present disclosure need not forma perfectly matched duplex with the single stranded nucleic acid, thougha perfect matched duplex is exemplary. According to one aspect,oligonucleotide probes as described herein form a stable hybrid withthat of the target sequence under stringent to moderately stringenthybridization and wash conditions. If it is expected that the probeswill be essentially completely complementary (i.e., about 99% orgreater) to the target sequence, stringent conditions will be used. Ifsome mismatching is expected, with the result that the probe will not becompletely complementary, the stringency of hybridization may belessened. Conditions which affect hybridization, and which selectagainst nonspecific binding are known in the art, and are described in,for example, Sambrook et al., (2001). Generally, lower saltconcentration and higher temperature increase the stringency of binding.For example, it is usually considered that stringent conditions areincubations in solutions which contain approximately 0.1×SSC, 0.1% SDS,at about 65° C. incubation/wash temperature, and moderately stringentconditions are incubations in solutions which contain approximately1-2×SSC, 0.1% SDS and about 50°−65° C. incubation/wash temperature. Lowstringency conditions are 2×SSC and about 30°−50° C.

The terms “stringency” or “stringent hybridization conditions” refer tohybridization conditions that affect the stability of hybrids, e.g.,temperature, salt concentration, pH, formamide concentration and thelike. These conditions are empirically optimized to maximize specificbinding and minimize non-specific binding of primer or probe to itstarget nucleic acid sequence. The terms as used include reference toexemplary conditions under which a probe or primer will hybridize to itstarget sequence, to a detectably greater degree than other sequences(e.g. at least 2-fold over background). Other such conditions may beappropriate. Stringent conditions are sequence dependent and will bedifferent in different circumstances. Longer sequences hybridizespecifically at higher temperatures. Generally, stringent conditions areselected to be about 5° C. lower than the thermal melting point (T_(m))for the specific sequence at a defined ionic strength and pH. The T_(m)is the temperature (under defined ionic strength and pH) at which 50% ofa complementary target sequence hybridizes to a perfectly matched probeor primer. Typically, stringent conditions will be those in which thesalt concentration is less than about 1.0 M Na⁺ ion, typically about0.01 to 1.0 M Na⁺ ion concentration (or other salts) at pH 7.0 to 8.3and the temperature is at least about 30° C. for short probes or primers(e.g. 10 to 50 nucleotides) and at least about 60° C. for long probes orprimers (e.g. greater than 50 nucleotides). Stringent conditions mayalso be achieved with the addition of destabilizing agents such asformamide.

Exemplary low stringent conditions or “conditions of reduced stringency”include hybridization with a buffer solution of 30% formamide, 1 M NaCl,1% SDS at 37° C. and a wash in 2×SSC at 40° C. Exemplary high stringencyconditions include hybridization in 50% formamide, 1M NaCl, 1% SDS at37° C., and a wash in 0.1×SSC at 60° C. Hybridization procedures arewell known in the art and are described by e.g. Ausubel et al., 1998 andSambrook et al., 2001. It is to be understood that any desiredstringency and/or conditions may be employed as desired.

Nucleic acid probes according to the present disclosure may be labeledor unlabeled. Certain nucleic acid probes may be directly labeled orindirectly labeled.

According to certain aspects, nucleic acid probes may include a primarynucleic acid sequence that is non-hybridizable to a target nucleic acidsequence in addition to the sequence of the probe that hybridizes to thetarget nucleic acid sequence. Exemplary primary nucleic acid sequencesor target non-hybridizing nucleic acid sequences include between about10 nucleotides to about 100 nucleotides, between about 10 nucleotides toabout 70 nucleotides, between about 15 nucleotides to about 50nucleotides, between about 20 nucleotides to about 60 nucleotides andall ranges and values in between whether overlapping or not. Accordingto certain aspects, the primary nucleic acid sequence is hybridizablewith one or more secondary nucleic acid sequences. According to certainaspects, the secondary nucleic acid sequence may include a label.According to this aspect, the nucleic acid probes are indirectly labeledas the secondary nucleic acid binds to the primary nucleic acid therebyindirectly labeling the probe which hybridizes to the target nucleicacid sequence. According to certain aspects, a plurality of nucleic acidprobes is provided with each having a common primary nucleic acidsequence. That is, the primary nucleic acid sequence is common to aplurality of nucleic acid probes, such that each nucleic acid probe inthe plurality has the same or substantially similar primary nucleic acidsequence. According to one aspect, the primary nucleic acid sequence isa single sequence species. In this manner, a plurality of commonsecondary nucleic acid sequences is provided which hybridize to theplurality of common primary nucleic acid sequences. That is, eachsecondary nucleic acid sequence has the same or substantially similarnucleic acid sequence. According to one exemplary embodiment, a singleprimary nucleic acid sequence is provided for each of the nucleic acidprobes in the plurality. Accordingly, only a single secondary nucleicacid sequence which is hybridizable to the primary nucleic acid sequenceneed be provided to label each of the nucleic acid probes. According tocertain aspects, the common secondary nucleic acid sequences may includea common label. According to this aspect, a plurality of nucleic acidprobes are provided having substantially diverse nucleic acid sequenceshybridizable to different target nucleic acid sequences and where theplurality of nucleic acid probes have common primary nucleic acidsequences. Accordingly, a common secondary nucleic acid sequence havinga label may be used to indirectly label each of the plurality of nucleicacid probes. According to this aspect, a single or common primarynucleic acid sequence and secondary nucleic acid sequence pair can beused to indirectly label diverse nucleic acid probe sequences. Such anembodiment is provided where a plurality of nucleic acid probes havingprimary nucleic acid sequences are commercially synthesized, such as onan array. Labeled secondary nucleic acid sequences can also becommercially synthesized so that they are hybridizable with the primarynucleic acid sequences. The nucleic acid probes may be combined with thelabeled secondary nucleic acids and one or more or a plurality of targetnucleic acid sequences under conditions such that the nucleic acid probeor probes hybridize to the target nucleic acid sequence or sequenceswhile the primary nucleic acid sequence is nonhybridizable to the targetnucleic acid sequence or sequences. A labeled secondary nucleic acidsequence hybridizes with a corresponding primary nucleic acid sequenceto indirectly label the nucleic acid probe, thereby labeling the targetnucleic acid sequence. According to one aspect, the nucleic acid probesmay be combined with the labeled secondary nucleic acids and one or moreor a plurality of target nucleic acid sequences together in a one potmethod. According to one aspect, the nucleic acid probes may be combinedwith the labeled secondary nucleic acids and one or more or a pluralityof target nucleic acid sequences sequentially, such as the nucleic acidprobes are combined with the target nucleic acid to form a mixture andthen the labeled secondary nucleic acid is combined with the mixture orthe nucleic acid probes are combined with the labeled secondary nucleicacids to form a mixture and then the target nucleic acid is combinedwith the mixture.

According to certain aspects, the primary nucleic acid sequence ismodifiable with one or more labels. According to this aspect, one ormore labels may be added to the primary nucleic acid sequence usingmethods known to those of skill in the art.

According to an additional embodiment, nucleic acid probes may include afirst half of a ligand-ligand binding pair, such as biotin-avidin. Suchnucleic acid probes may or may not include a primary nucleic acidsequence. The first half of a ligand-ligand binding pair may be attacheddirectly to the nucleic acid probe. According to certain aspects, asecond half of the ligand-ligand binding pair may include a label.Accordingly, the nucleic acid probe may be indirectly labeled by the useof a ligand-ligand binding pair. According to certain aspects, a commonligand-ligand binding pair may be used with a plurality of nucleic acidprobes of different nucleic acid sequences. Accordingly, a singlespecies of ligand-ligand binding pair may be used to indirectly label aplurality of different nucleic acid probe sequences. The commonligand-ligand binding pair may include a common label or a plurality ofcommon ligand-ligand binding pairs may be labeled with different labels.Accordingly, a plurality of nucleic acid probes of different nucleicacid sequences may be labeled with a single species of label using asingle species of a ligand-ligand binding pair.

According to one aspect, the primary nucleic acid sequences may includeone or more subsequences that are hybridizable with one or moredifferent secondary nucleic sequences. The one or more secondary nucleicacid sequences may include one or more subsequences that hybridize withone or more tertiary nucleic acid sequences, and so on. Each of theprimary nucleic acid sequences, the secondary nucleic acid sequences,the tertiary nucleic acid sequences and so on may be directly labeledwith a label or may be indirectly labeled with a label. In this manner,an exponential labeling of the nucleic acid probe can be achieved.

Labels

A label according to the present disclosure includes a functional moietydirectly or indirectly attached or conjugated to a nucleic acid whichprovides a desired function. According to certain aspects, a label maybe used for detection. Detectable labels or moieties are known to thoseof skill in the art. According to certain aspects, a label may be usedto retrieve a particular molecule. Retrievable labels or moieties areknown to those of skill in the art. According to certain aspects, alabel may be used to target a particular molecule to a target nucleicacid of interest for a desired function. Targeting labels or moietiesare known to those of skill in the art. According to certain aspects, alabel may be used to react with a target nucleic acid of interest.Reactive labels or moieties are known to those of skill in the art.According to certain aspects, a label may be an antibody, ligand,hapten, radioisotope, therapeutic agent and the like.

As used herein, the term “retrievable moiety” refers to a moiety that ispresent in or attached to a polynucleotide that can be used to retrievea desired molecule or factors bound to a desired molecule (e.g., one ormore factors bound to a targeting moiety). As used herein, the term“retrievable label” refers to a label that is attached to apolynucleotide (e.g., an Oligopaint) and can, optionally, be used tospecifically and/or nonspecifically bind a target protein, peptide, DNAsequence, RNA sequence, carbohydrate or the like at or near thenucleotide sequence to which one or more Oligopaints have hybridized. Incertain aspects, target proteins include, but are not limited to,proteins that are involved with gene regulation such as, e.g., proteinsassociated with chromatin (See, e.g., Dejardin and Kingston (2009) Cell136:175), proteins that regulate (upregulate or downregulate)methylation, proteins that regulate (upregulate or downregulate) histoneacetylation, proteins that regulate (upregulate or downregulate)transcription, proteins that regulate (upregulate or downregulate)post-transcriptional regulation, proteins that regulate (upregulate ordownregulate) RNA transport, proteins that regulate (upregulate ordownregulate) mRNA degradation, proteins that regulate (upregulate ordownregulate) translation, proteins that regulate (upregulate ordownregulate) post-translational modifications and the like.

As used herein, the term “targeting moiety” refers to a moiety that ispresent in or attached to a polynucleotide that can be used tospecifically and/or nonspecifically bind one or more factors thatassociate with, modify or otherwise interact with a nucleic acidsequence of interest (e.g., DNA (e.g., nuclear, mitochondrial,transfected and the like) and/or RNA), including, but not limited to, aprotein, a peptide, a DNA sequence, an RNA sequence, a carbohydrate, alipid, a chemical moiety or the like at or near the nucleotide sequenceof interest to which the polynucleotide has hybridized. In certainaspects, factors that associate with a nucleic acid sequence of interestinclude, but are not limited to histone proteins (e.g., H1, H2A, H2B,H3, H4 and the like, including monomers and oligomers (e.g., dimers,tetramers, octamers and the like)) scaffold proteins, transcriptionfactors, DNA binding proteins, DNA repair factors, DNA modificationproteins (e.g., acetylases, methylases and the like).

In other aspects, factors that associate with, modify or otherwiseinteract with a nucleic acid sequence of interest are proteinsincluding, but not limited to, proteins that are involved with generegulation such as, e.g., proteins associated with chromatin (See, e.g.,Dejardin and Kingston (2009) Cell 136:175), proteins that regulate(upregulate or downregulate) methylation, proteins that regulate(upregulate or downregulate) acetylation, proteins that regulate(upregulate or downregulate) histone acetylation, proteins that regulate(upregulate or downregulate) transcription, proteins that regulate(upregulate or downregulate) post-transcriptional regulation, proteinsthat regulate (upregulate or downregulate) RNA transport, proteins thatregulate (upregulate or downregulate) mRNA degradation, proteins thatregulate (upregulate or downregulate) translation, proteins thatregulate (upregulate or downregulate) post-translational modificationsand the like.

In certain aspects, a targeting and/or retrievable moiety isactivatable. As used herein, the term “activatable” refers to atargeting and/or retrievable moiety that is inert (i.e., does not bind atarget) until activated (e.g., by exposure of the activatable, targetingand/or retrievable moiety to light, heat, one or more chemical compoundsor the like). In other aspects, a targeting and/or retrievable moietycan bind one or more targets without the need for activation of thetargeting and/or retrievable moiety. Exemplary methods for attachingproteins, lipids, carbohydrates, nucleic acids and the like are known tothose of skill in the art. In certain aspects, a targeting moiety can bea non-targeting moiety that is cross-linked or otherwise modified tobind one or more factors that associate with, modify or otherwiseinteract with a nucleic acid sequence.

In certain exemplary embodiments, a targeting moiety, a retrievablemoiety and/or polynucleotide has a detectable label bound thereto. Asused herein, the term “detectable label” refers to a label that can beused to identify a target (e.g., a factor associated with a nucleic acidsequence of interest, a chromosome or a sub-chromosomal region).Typically, a detectable label is attached to the 3′- or 5′-end of apolynucleotide. Alternatively, a detectable label is attached to aninternal portion of an oligonucleotide. Detectable labels may varywidely in size and compositions; the following references provideguidance for selecting oligonucleotide tags appropriate for particularembodiments: Brenner, U.S. Pat. No. 5,635,400; Brenner et al., Proc.Natl. Acad. Sci., 97: 1665; Shoemaker et al. (1996) Nature Genetics,14:450; Morris et al., EP Patent Pub. 0799897A1; Wallace, U.S. Pat. No.5,981,179; and the like.

Methods for incorporating detectable labels into nucleic acid probes arewell known. Typically, detectable labels (e.g., as hapten- orfluorochrome-conjugated deoxyribonucleotides) are incorporated into anucleic acid, such as a nucleic acid probe during a polymerization oramplification step, e.g., by PCR, nick translation, random primerlabeling, terminal transferase tailing (e.g., one or more labels can beadded after cleavage of the primer sequence), and others (see Ausubel etal., 1997, Current Protocols In Molecular Biology, Greene Publishing andWiley-Interscience, New York).

In certain aspects, a suitable targeting moiety, retrievable moiety ordetectable label includes, but is not limited to, a capture moiety suchas a hydrophobic compound, an oligonucleotide, an antibody or fragmentof an antibody, a protein, a peptide, a chemical cross-linker, anintercalator, a molecular cage (e.g., within a cage or other structure,e.g., protein cages, fullerene cages, zeolite cages, photon cages, andthe like), or one or more elements of a capture pair, e.g.,biotin-avidin, biotin-streptavidin, NHS-ester and the like, a thioetherlinkage, static charge interactions, van der Waals forces and the like(See, e.g., Holtke et al., U.S. Pat. Nos. 5,344,757; 5,702,888; and U.S.Pat. No. 5,354,657; Huber et al., U.S. Pat. No. 5,198,537; Miyoshi, U.S.Pat. No. 4,849,336; Misiura and Gait, PCT publication WO 91/17160). Incertain aspects, a suitable targeting label, retrievable label ordetectable label is an enzyme (e.g., a methylase and/or a cleavingenzyme). In one aspect, an antibody specific against the enzyme can beused to retrieve or detect the enzyme and accordingly, retrieve ordetect an oligonucleotide sequence or factor attached to the enzyme. Inanother aspect, an antibody specific against the enzyme can be used toretrieve or detect the enzyme and, after stringent washes, retrieve ordetect a factor or first oligonucleotide sequence that is hybridized toa second oligonucleotide sequence having the enzyme attached thereto.

Biotin, or a derivative thereof, may be used as an oligonucleotide label(e.g., as a targeting moiety, retrievable moiety and/or a detectablelabel), and subsequently bound by a avidin/streptavidin derivative(e.g., detectably labelled, e.g., phycoerythrin-conjugatedstreptavidin), or an anti-biotin antibody (e.g., a detectably labelledantibody). Digoxigenin may be incorporated as a label and subsequentlybound by a detectably labelled anti-digoxigenin antibody (e.g., adetectably labelled antibody, e.g., fluoresceinated anti-digoxigenin).An aminoallyl-dUTP residue may be incorporated into an oligonucleotideand subsequently coupled to an N-hydroxy succinimide (NHS) derivatizedfluorescent dye. In general, any member of a conjugate pair may beincorporated into a retrievable moiety and/or a detectable labelprovided that a detectably labelled conjugate partner can be bound topermit detection. As used herein, the term antibody refers to anantibody molecule of any class, or any sub-fragment thereof, such as anFab.

Other suitable labels (targeting moieties, retrievable moieties and/ordetectable labels) include, but are not limited to, fluorescein (FAM),digoxigenin, dinitrophenol (DNP), dansyl, biotin, bromodeoxyuridine(BrdU), hexahistidine (6×His), phosphor-amino acids (e.g. P-tyr, P-ser,P-thr) and the like. In one embodiment the following hapten/antibodypairs are used for reaction, retrieval and/or detection:biotin/α-biotin, digoxigenin/a-digoxigenin, dinitrophenol (DNP)/α-DNP,5-Carboxyfluorescein (FAM)/α-FAM.

Additional suitable labels (targeting moieties, retrievable moietiesand/or detectable labels) include, but are not limited to, chemicalcross-linking agents. Cross-linking agents typically contain at leasttwo reactive groups that are reactive towards numerous groups,including, but not limited to, sulfhydryls and amines, and createchemical covalent bonds between two or more molecules. Functional groupsthat can be targeted with cross-linking agents include, but are notlimited to, primary amines, carboxyls, sulfhydryls, carbohydrates andcarboxylic acids. Protein molecules have many of these functional groupsand therefore proteins and peptides can be readily conjugated usingcross-linking agents. Cross-linking agents are well known in the art andare commercially available (Thermo Scientific (Rockford, Ill.)).

A detectable moiety, label or reporter can be used to detect a nucleicacid or nucleic acid probe as described herein. Oligonucleotide probesor nucleic acid probes described herein can be labeled in a variety ofways, including the direct or indirect attachment of a detectable moietysuch as a fluorescent moiety, hapten, colorimetric moiety and the like.A location where a label may be attached is referred to herein as alabel addition site or detectable moiety addition site and may include anucleotide to which the label is capable of being attached. One of skillin the art can consult references directed to labeling DNA. Examples ofdetectable moieties include various radioactive moieties, enzymes,prosthetic groups, fluorescent markers, luminescent markers,bioluminescent markers, metal particles, protein-protein binding pairs,protein-antibody binding pairs and the like. Examples of fluorescentmoieties include, but are not limited to, yellow fluorescent protein(YFP), green fluorescence protein (GFP), cyan fluorescence protein(CFP), umbelliferone, fluorescein, fluorescein isothiocyanate,rhodamine, dichlorotriazinylamine fluorescein, cyanines, dansylchloride, phycocyanin, phycoerythrin and the like. Examples ofbioluminescent markers include, but are not limited to, luciferase(e.g., bacterial, firefly, click beetle and the like), luciferin,aequorin and the like. Examples of enzyme systems having visuallydetectable signals include, but are not limited to, galactosidases,glucorinidases, phosphatases, peroxidases, cholinesterases and the like.Identifiable markers also include radioactive compounds such as ¹²⁵I,³⁵S, ¹⁴C, or ³H. Identifiable markers are commercially available from avariety of sources.

Fluorescent labels and their attachment to nucleotides and/oroligonucleotides are described in many reviews, including Haugland,Handbook of Fluorescent Probes and Research Chemicals, Ninth Edition(Molecular Probes, Inc., Eugene, 2002); Keller and Manak, DNA Probes,2nd Edition (Stockton Press, New York, 1993); Eckstein, editor,Oligonucleotides and Analogues: A Practical Approach (IRL Press, Oxford,1991); and Wetmur, Critical Reviews in Biochemistry and MolecularBiology, 26:227-259 (1991). Particular methodologies applicable to theinvention are disclosed in the following sample of references: U.S. Pat.Nos. 4,757,141, 5,151,507 and 5,091,519. In one aspect, one or morefluorescent dyes are used as labels for labeled target sequences, e.g.,as disclosed by U.S. Pat. No. 5,188,934 (4,7-dichlorofluorescein dyes);U.S. Pat. No. 5,366,860 (spectrally resolvable rhodamine dyes); U.S.Pat. No. 5,847,162 (4,7-dichlororhodamine dyes); U.S. Pat. No. 4,318,846(ether-substituted fluorescein dyes); U.S. Pat. No. 5,800,996 (energytransfer dyes); Lee et al.; U.S. Pat. No. 5,066,580 (xanthine dyes);U.S. Pat. No. 5,688,648 (energy transfer dyes); and the like. Labelingcan also be carried out with quantum dots, as disclosed in the followingpatents and patent publications: U.S. Pat. Nos. 6,322,901, 6,576,291,6,423,551, 6,251,303, 6,319,426, 6,426,513, 6,444,143, 5,990,479,6,207,392, 2002/0045045 and 2003/0017264. As used herein, the term“fluorescent label” includes a signaling moiety that conveys informationthrough the fluorescent absorption and/or emission properties of one ormore molecules. Such fluorescent properties include fluorescenceintensity, fluorescence lifetime, emission spectrum characteristics,energy transfer, and the like.

Commercially available fluorescent nucleotide analogues readilyincorporated into nucleotide and/or oligonucleotide sequences include,but are not limited to, Cy3-dCTP, Cy3-dUTP, Cy5-dCTP, Cy5-dUTP (AmershamBiosciences, Piscataway, N.J.), fluorescein-12-dUTP,tetramethylrhodamine-6-dUTP, TEXAS RED™-5-dUTP, CASCADE BLUE™-7-dUTP,BODIPY TMFL-14-dUTP, BODIPY TMR-14-dUTP, BODIPY TMTR-14-dUTP, RHODAMINEGREEN™-5-dUTP, OREGON GREENR™ 488-5-dUTP, TEXAS RED™-12-dUTP, BODIPY™630/650-14-dUTP, BODIPY™ 650/665-14-dUTP, ALEXA FLUOR™ 488-5-dUTP, ALEXAFLUOR™ 532-5-dUTP, ALEXA FLUOR™ 568-5-dUTP, ALEXA FLUOR™ 594-5-dUTP,ALEXA FLUOR™ 546-14-dUTP, fluorescein-12-UTP,tetramethylrhodamine-6-UTP, TEXAS RED™-5-UTP, mCherry, CASCADEBLUE™-7-UTP, BODIPY™ FL-14-UTP, BODIPY TMR-14-UTP, BODIPY™ TR-14-UTP,RHODAMINE GREEN™-5-UTP, ALEXA FLUOR™ 488-5-UTP, LEXA FLUOR™ 546-14-UTP(Molecular Probes, Inc. Eugene, Oreg.) and the like. Alternatively, theabove fluorophores and those mentioned herein may be added duringoligonucleotide synthesis using for example phosphoroamidite or NHSchemistry. Protocols are known in the art for custom synthesis ofnucleotides having other fluorophores (See, Henegariu et al. (2000)Nature Biotechnol. 18:345). 2-Aminopurine is a fluorescent base that canbe incorporated directly in the oligonucleotide sequence during itssynthesis. Nucleic acid could also be stained, a priori, with anintercalating dye such as DAPI, YOYO-1, ethidium bromide, cyanine dyes(e.g. SYBR Green) and the like.

Other fluorophores available for post-synthetic attachment include, butare not limited to, ALEXA FLUOR™ 350, ALEXA FLUOR™ 405, ALEXA FLUOR™430, ALEXA FLUOR™ 532, ALEXA FLUOR™ 546, ALEXA FLUOR™ 568, ALEXA FLUOR™594, ALEXA FLUOR™ 647, BODIPY 493/503, BODIPY FL, BODIPY R6G, BODIPY530/550, BODIPY TMR, BODIPY 558/568, BODIPY 558/568, BODIPY 564/570,BODIPY 576/589, BODIPY 581/591, BODIPY TR, BODIPY 630/650, BODIPY650/665, Cascade Blue, Cascade Yellow, Dansyl, lissamine rhodamine B,Marina Blue, Oregon Green 488, Oregon Green 514, Pacific Blue, PacificOrange, rhodamine 6G, rhodamine green, rhodamine red, tetramethylrhodamine, Texas Red (available from Molecular Probes, Inc., Eugene,Oreg.), Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7 (Amersham Biosciences,Piscataway, N.J.) and the like. FRET tandem fluorophores may also beused, including, but not limited to, PerCP-Cy5.5, PE-Cy5, PE-Cy5.5,PE-Cy7, PE-Texas Red, APC-Cy7, PE-Alexa dyes (610, 647, 680), APC-Alexadyes and the like.

FRET tandem fluorophores may also be used, such as PerCP-Cy5.5, PE-Cy5,PE-Cy5.5, PE-Cy7, PE-Texas Red, and APC-Cy7; also, PE-Alexa dyes (610,647, 680) and APC-Alexa dyes.

Metallic silver or gold particles may be used to enhance signal fromfluorescently labeled nucleotide and/or oligonucleotide sequences(Lakowicz et al. (2003) BioTechniques 34:62).

Biotin, or a derivative thereof, may also be used as a label on anucleotide and/or an oligonucleotide sequence, and subsequently bound bya detectably labeled avidin/streptavidin derivative (e.g.phycoerythrin-conjugated streptavidin), or a detectably labeledanti-biotin antibody. Biotin/avidin is an example of a ligand-ligandbinding pair. An antibody/antigen binging pair may also be used withmethods described herein. Other ligand-ligand binding pairs or conjugatebinding pairs are well known to those of skill in the art. Digoxigeninmay be incorporated as a label and subsequently bound by a detectablylabeled anti-digoxigenin antibody (e.g. fluoresceinatedanti-digoxigenin). An aminoallyl-dUTP or aminohexylacrylamide-dCTPresidue may be incorporated into an oligonucleotide sequence andsubsequently coupled to an N-hydroxy succinimide (NHS) derivatizedfluorescent dye. In general, any member of a conjugate pair may beincorporated into a detection oligonucleotide provided that a detectablylabeled conjugate partner can be bound to permit detection. As usedherein, the term antibody refers to an antibody molecule of any class,or any sub-fragment thereof, such as an Fab.

Other suitable labels for an oligonucleotide sequence may includefluorescein (FAM, FITC), digoxigenin, dinitrophenol (DNP), dansyl,biotin, bromodeoxyuridine (BrdU), hexahistidine (6×His), phosphor-aminoacids (e.g. P-tyr, P-ser, P-thr) and the like. In one embodiment thefollowing hapten/antibody pairs are used for detection, in which each ofthe antibodies is derivatized with a detectable label: biotin/α-biotin,digoxigenin/α-digoxigenin, dinitrophenol (DNP)/α-DNP,5-Carboxyfluorescein (FAM)/α-FAM.

In certain exemplary embodiments, a nucleotide and/or an oligonucleotidesequence can be indirectly labeled, especially with a hapten that isthen bound by a capture agent, e.g., as disclosed in U.S. Pat. Nos.5,344,757, 5,702,888, 5,354,657, 5,198,537 and 4,849,336, PCTpublication WO 91/17160 and the like. Many different hapten-captureagent pairs are available for use. Exemplary haptens include, but arenot limited to, biotin, des-biotin and other derivatives, dinitrophenol,dansyl, fluorescein, CY5, digoxigenin and the like. For biotin, acapture agent may be avidin, streptavidin, or antibodies. Antibodies maybe used as capture agents for the other haptens (many dye-antibody pairsbeing commercially available, e.g., Molecular Probes, Eugene, Oreg.).

According to certain aspects, detectable moieties described herein arespectrally resolvable. “Spectrally resolvable” in reference to aplurality of fluorescent labels means that the fluorescent emissionbands of the labels are sufficiently distinct, i.e., sufficientlynon-overlapping, that molecular tags to which the respective labels areattached can be distinguished on the basis of the fluorescent signalgenerated by the respective labels by standard photodetection systems,e.g., employing a system of band pass filters and photomultiplier tubes,or the like, as exemplified by the systems described in U.S. Pat. Nos.4,230,558; 4,811,218, or the like, or in Wheeless et al., pgs. 21-76, inFlow Cytometry: Instrumentation and Data Analysis (Academic Press, NewYork, 1985). In one aspect, spectrally resolvable organic dyes, such asfluorescein, rhodamine, and the like, means that wavelength emissionmaxima are spaced at least 20 nm apart, and in another aspect, at least40 nm apart. In another aspect, chelated lanthanide compounds, quantumdots, and the like, spectrally resolvable means that wavelength emissionmaxima are spaced at least 10 nm apart, and in a further aspect, atleast 15 nm apart.

In certain embodiments, the detectable moieties can provide higherdetectability when used with an electron microscope, compared withcommon nucleic acids. Moieties with higher detectability are often inthe group of metals and organometals, such as mercuric acetate, platinumdimethylsulfoxide, several metal-bipyridyl complexes (e.g. osmium-bipy,ruthenium-bipy, platinum-bipy). While some of these moieties can readilystain nucleic acids specifically, linkers can also be used to attachthese moieties to a nucleic acid. Such linkers added to nucleotidesduring synthesis are acrydite- and a thiol-modified entities, aminereactive groups, and azide and alkyne groups for performing clickchemistry. Some nucleic acid analogs are also more detectable such asgamma-adenosine-thiotriphosphate, iododeoxycytidine-triphosphate, andmetallonucleosides in general (see Dale et al., Proc. Nat. Acad. Sci.USA, Vol. 70, No. 8, pp. 2238-2242 (1973)). The modified nucleotides areadded during synthesis. Synthesis may refer by example to solid supportsynthesis of oligonucleotides. In this case, modified nucleic acids,which can be a nucleic acid analog, or a nucleic acid modified with adetectable moiety, or with an attachment chemistry linker, are added oneafter each other to the nucleic acid fragments being formed on the solidsupport, with synthesis by phosphoramidite being the most popularmethod. Synthesis may also refer to the process performed by apolymerase while it synthesizes the complementary strands of a nucleicacid template. Certain DNA polymerases are capable of using andincorporating nucleic acids analogs, or modified nucleic acids, eithermodified with a detectable moiety or an attachment chemistry linker tothe complementary nucleic acid template.

Detection method(s) used will depend on the particular detectable labelsused in the reactive labels, retrievable labels and/or detectablelabels. In certain exemplary embodiments, target nucleic acids such aschromosomes and sub-chromosomal regions of chromosomes during variousphases of the cell cycle including, but not limited to, interphase,preprophase, prophase, prometaphase, metaphase, anaphase, telophase andcytokinesis, having one or more reactive labels, retrievable labels, ordetectable labels bound thereto by way of the probes described hereinmay be selected for and/or screened for using a microscope, aspectrophotometer, a tube luminometer or plate luminometer, x-ray film,a scintillator, a fluorescence activated cell sorting (FACS) apparatus,a microfluidics apparatus or the like.

When fluorescently labeled targeting moieties, retrievable moieties, ordetectable labels are used, fluorescence photomicroscopy can be used todetect and record the results of in situ hybridization using routinemethods known in the art. Alternatively, digital (computer implemented)fluorescence microscopy with image-processing capability may be used.Two well-known systems for imaging FISH of chromosomes having multiplecolored labels bound thereto include multiplex-FISH (M-FISH) andspectral karyotyping (SKY). See Schrock et al. (1996) Science 273:494;Roberts et al. (1999) Genes Chrom. Cancer 25:241; Fransz et al. (2002)Proc. Natl. Acad. Sci. USA 99:14584; Bayani et al. (2004) Curr.Protocol. Cell Biol. 22.5.1-22.5.25; Danilova et al. (2008) Chromosoma117:345; U.S. Pat. No. 6,066,459; and FISH TAG™ DNA Multicolor Kitinstructions (Molecular probes) for a review of methods for paintingchromosomes and detecting painted chromosomes.

In certain exemplary embodiments, images of fluorescently labeledchromosomes are detected and recorded using a computerized imagingsystem such as the Applied Imaging Corporation CytoVision System(Applied Imaging Corporation, Santa Clara, Calif.) with modifications(e.g., software, Chroma 84000 filter set, and an enhanced filter wheel).Other suitable systems include a computerized imaging system using acooled CCD camera (Photometrics, NU200 series equipped with Kodak KAF1400 CCD) coupled to a Zeiss Axiophot microscope, with images processedas described by Ried et al. (1992) Proc. Natl. Acad. Sci. USA 89:1388).Other suitable imaging and analysis systems are described by Schrock etal., supra; and Speicher et al., supra.

In situ hybridization methods using probes described herein can beperformed on a variety of biological or clinical samples, in cells thatare in any (or all) stage(s) of the cell cycle (e.g., mitosis, meiosis,interphase, G0, G1, S and/or G2). Examples include all types of cellculture, animal or plant tissue, peripheral blood lymphocytes, buccalsmears, touch preparations prepared from uncultured primary tumors,cancer cells, bone marrow, cells obtained from biopsy or cells in bodilyfluids (e.g., blood, urine, sputum and the like), cells from amnioticfluid, cells from maternal blood (e.g., fetal cells), cells from testisand ovary, and the like. Samples are prepared for assays of theinvention using conventional techniques, which typically depend on thesource from which a sample or specimen is taken. These examples are notto be construed as limiting the sample types applicable to the methodsand/or compositions described herein.

In certain exemplary embodiments, probes include multiplechromosome-specific probes, which are differentially labeled (i.e., atleast two of the chromosome-specific probes are differently labeled).Various approaches to multi-color chromosome painting have beendescribed in the art and can be adapted to the present inventionfollowing the guidance provided herein. Examples of such differentiallabeling (“multicolor FISH”) include those described by Schrock et al.(1996) Science 273:494, and Speicher et al. (1996) Nature Genet.12:368). Schrock et al. describes a spectral imaging method, in whichepifluorescence filter sets and computer software is used to detect anddiscriminate between multiple differently labeled DNA probes hybridizedsimultaneously to a target chromosome set. Speicher et al. describesusing different combinations of 5 fluorochromes to label each of thehuman chromosomes (or chromosome arms) in a 27-color FISH termed“combinatorial multifluor FISH”). Other suitable methods may also beused (see, e.g., Ried et al., 1992, Proc. Natl. Acad. Sci. USA89:1388-92).

Hybridization of the labeled probes described herein to targetchromosomes sequences can be accomplished by standard in situhybridization (ISH) techniques (see, e.g., Gall and Pardue (1981) Meth.Enzymol. 21:470; Henderson (1982) Int. Review of Cytology 76:1).Generally, ISH comprises the following major steps: (1) fixation of thebiological structure to be analyzed (e.g., a chromosome spread), (2)pre-hybridization treatment of the biological structure to increaseaccessibility of target DNA (e.g., denaturation with heat or alkali),(3) optional pre-hybridization treatment to reduce nonspecific binding(e.g., by blocking the hybridization capacity of repetitive sequences),(4) hybridization of the mixture of nucleic acids to the nucleic acid inthe biological structure or tissue; (5) post-hybridization washes toremove nucleic acid fragments not bound in the hybridization and (6)detection of the hybridized labelled oligonucleotides (e.g., hybridizedOligopaints). The reagents used in each of these steps and theirconditions of use vary depending on the particular situation and whethertheir use is required with any particular probes. Hybridizationconditions are also described in U.S. Pat. No. 5,447,841. It will beappreciated that numerous variations of in situ hybridization protocolsand conditions are known and may be used in conjunction with the presentinvention by practitioners following the guidance provided herein.

Ligation

Ligation methods are known to those of skill in the art and includethose disclosed in Metzker, Nature Reviews/Genetics, Vol. 11, pp. 31-46(2010) hereby incorporated by reference in its entirety for allpurposes.

Methods of hybridizing and ligating oligonucleotide probes to a singlestranded template nucleic acid are known to those of skill in the art.“Hybridization” refers to the process in which two single-strandedpolynucleotides bind non-covalently to form a stable double-strandedpolynucleotide. The term “hybridization” may also refer totriple-stranded hybridization. The resulting (usually) double-strandedpolynucleotide is a “hybrid” or “duplex.” “Hybridization conditions”will typically include salt concentrations of less than about 1 M, moreusually less than about 500 mM and even more usually less than about 200mM. Hybridization temperatures can be as low as 5° C., but are typicallygreater than 22° C., more typically greater than about 30° C., and oftenin excess of about 37° C. Hybridizations are usually performed understringent conditions, i.e., conditions under which a probe willhybridize to its target subsequence. Stringent conditions aresequence-dependent and are different in different circumstances. Longerfragments may require higher hybridization temperatures for specifichybridization. As other factors may affect the stringency ofhybridization, including base composition and length of thecomplementary strands, presence of organic solvents and extent of basemismatching, the combination of parameters is more important than theabsolute measure of any one alone. Generally, stringent conditions areselected to be about 5° C. lower than the T_(m) for the specificsequence at s defined ionic strength and pH. Exemplary stringentconditions include salt concentration of at least 0.01 M to no more than1 M Na ion concentration (or other salts) at a pH 7.0 to 8.3 and atemperature of at least 25° C. For example, conditions of 5×SSPE (750 mMNaCl, 50 mM Na phosphate, 5 mM EDTA, pH 7.4) and a temperature of 25-30°C. are suitable for allele-specific probe hybridizations. For stringentconditions, see for example, Sambrook, Fritsche and Maniatis, MolecularCloning A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press (1989) andAnderson Nucleic Acid Hybridization, 1^(st) Ed., BIOS ScientificPublishers Limited (1999). “Hybridizing specifically to” or“specifically hybridizing to” or like expressions refer to the binding,duplexing, or hybridizing of a molecule substantially to or only to aparticular nucleotide sequence or sequences under stringent conditionswhen that sequence is present in a complex mixture (e.g., totalcellular) DNA or RNA.

Ligation can be accomplished either enzymatically or chemically.“Ligation” means to form a covalent bond or linkage between the terminiof two or more nucleic acids, e.g., oligonucleotides and/orpolynucleotides, in a template-driven reaction. The nature of the bondor linkage may vary widely and the ligation may be carried outenzymatically or chemically. As used herein, ligations are usuallycarried out enzymatically to form a phosphodiester linkage between a 5′carbon of a terminal nucleotide of one oligonucleotide with 3′ carbon ofanother oligonucleotide. A variety of template-driven ligation reactionsare described in the following references: Whitely et al., U.S. Pat. No.4,883,750; Letsinger et al., U.S. Pat. No. 5,476,930; Fung et al., U.S.Pat. No. 5,593,826; Kool, U.S. Pat. No. 5,426,180; Landegren et al.,U.S. Pat. No. 5,871,921; Xu and Kool (1999) Nucl. Acids Res. 27:875;Higgins et al., Meth. in Enzymol. (1979) 68:50; Engler et al. (1982) TheEnzymes, 15:3 (1982); and Namsaraev, U.S. Patent Pub. 2004/0110213.

Chemical ligation methods are disclosed in Ferris et al., Nucleosides &Nucleotides, 8: 407-414 (1989) and Shabarova et al., Nucleic Acidsresearch, 19: 4247-4251 (1991). Enzymatic ligation utilizes a ligase.Many ligases are known to those of skill in the art as referenced inLehman, Science, 186: 790-797 (1974); Engler et al., DNA ligases, pages3-30 in Boyer, editor, The Enzymes, Vol. 15B (Academic Press, New York,1982); and the like. Exemplary ligases include T4 DNA ligase, T7 DNAligase, E. coli DNA ligase, Taq ligase, Pfu ligase and the like. Certainprotocols for using ligases are disclosed by the manufacturer and alsoin Sambrook, Molecular Cloning: A Laboratory manual, 2^(nd) Edition(Cold Spring Harbor Laboratory, New York, 1989); barany, PCR Methods andApplications, 1:5-16 (1991); Marsh et al., Strategies, 5:73-76 (1992).

Methods described herein using chain extension over a sequencedifference to differentially label a sequence difference can alsoinclude ligation methods described herein to differentially label asequence difference by substituting a ligation method for a chainextension method.

Amplification

The expression “amplification” or “amplifying” refers to a process bywhich extra or multiple copies of a particular polynucleotide areformed. Amplification includes methods such as PCR, ligationamplification (or ligase chain reaction, LCR) and amplification methods.These methods are known and widely practiced in the art. See, e.g., U.S.Pat. Nos. 4,683,195 and 4,683,202 and Innis et al., “PCR protocols: aguide to method and applications” Academic Press, Incorporated (1990)(for PCR); and Wu et al. (1989) Genomics 4:560-569 (for LCR). Ingeneral, the PCR procedure describes a method of gene amplificationwhich is comprised of (i) sequence-specific hybridization of primers tospecific genes within a DNA sample (or library), (ii) subsequentamplification involving multiple rounds of annealing, elongation, anddenaturation using a DNA polymerase, and (iii) screening the PCRproducts for a band of the correct size. The primers used areoligonucleotides of sufficient length and appropriate sequence toprovide initiation of polymerization, i.e. each primer is specificallydesigned to be complementary to each strand of the genomic locus to beamplified.

Reagents and hardware for conducting amplification reaction arecommercially available. Primers useful to amplify sequences from aparticular gene region are preferably complementary to, and hybridizespecifically to sequences in the target region or in its flankingregions and can he prepared using the polynucleotide sequences providedherein. Nucleic acid sequences generated by amplification can besequenced directly.

The term “amplification reagents” refers to those reagents(deoxyribonucleotide triphosphates, buffer, etc.), needed foramplification except for primers, nucleic acid template, and theamplification enzyme. Typically, amplification reagents along with otherreaction components are placed and contained in a reaction vessel (testtube, microwell, etc.). Amplification methods include PCR methods knownto those of skill in the art and also include rolling circleamplification (Blanco et al., J. Biol. Chem., 264, 8935-8940, 1989),hyperbranched rolling circle amplification (Lizard et al., Nat.Genetics, 19, 225-232, 1998), and loop-mediated isothermal amplification(Notomi et al., Nuc. Acids Res., 28, e63, 2000) each of which are herebyincorporated by reference in their entireties.

Methods for in situ amplification useful for the methods describedherein are described in Nilsson, Histochem Cell Biol (2006) 126:159-164;Diep et al., Nature Methods, Vol. 9, No. 3, pp. 270-274 (2012); Wang etal., Cancer Genetics, Vol. 205, Issues 7-8, pp. 341-355 (2012); Larssonet al., Nature Methods, Vol. 7, No. 5, pp. 395-400 (2010) and Zhang etal., Nature Genetics, Vol. 38, No. 3, pp. 382-387 (2006) each of whichare hereby incorporated by reference in their entireties for allpurposes.

Example I Extending Across SNPs

FIG. 1 is a schematic representation of a maternal homolog and apaternal homolog. SNPs are identified that distinguish one homolog fromanother. As can be seen in FIG. 1, the maternal homolog and the paternalhomolog are shown as being double stranded. For purposes of ease, thefollowing figures show only a single strand.

FIG. 2 is a schematic representation of a first SNP having a firstnucleotide type A on the maternal homolog. On the SNPs identified inFIG. 1, only those SNPs that put a particular base on only one of thehomologs are selected. For example, SNPs of nucleotide type A areidentified on the maternal homolog.

As depicted in FIG. 3, in order to label the paternal homolog, SNPs areselected on the paternal homolog that are not on the maternal homolog.For example, SNPs of nucleotide type G are selected.

As depicted in FIG. 4, in order to augment signal from a homolog, anadditional set of SNPs can be selected for the maternal homolog thathave not already been selected. For example, SNPs of nucleotide type Tcan be selected for the maternal homolog. Similarly, to enhance signalsfrom the paternal homolog, SNPs of nucleotide type C can be selected.

As depicted in FIG. 5, augmenting the number of SNPs to be used may meanthat some SNPs selected will no longer be appropriate because thecorrect nucleotide in the opposite homolog has been selected as a targetfor labeling.

As shown in FIG. 6, the usable nucleotides is reduced.

As shown in FIG. 7, oligonucleotide probes are hybridized tocomplementary sequences directly upstream of selected SNPs. Labeledbases and a suitable polymerase are added for extension of the probeacross the SNP.

As shown in FIG. 8, the probe is extended across the SNP using labels sothat the homologs become differentially labeled. For example, aCy3-labeled (red) T and a Cy3-labeled A can be used for the maternalhomolog. A Cy5-labeled (green) G and a Cy5-labeled C can be used for thepaternal homolog. This results in differential labeling of the homologs.

As shown in FIG. 9, a modified nucleotide, such as a non-naturallyoccurring nucleotide being complementary to a SNP can be used to coverthe SNP. Modified nucleotides may include biotinylated bases, bases usedfor chain extension and bases conjugated to fluors. According to thisaspect the modified bases which include non-naturally occurring basesare not present elsewhere in the genome. The term non-naturallynucleotide may also refer to a modified nucleotide.

As shown in FIG. 10, the quality of “n”, i.e. the moiety making the basenon-natural, can differ from base to base. The non-naturally occurringnucleotides corresponding to the maternal homolog share a feature “m”which is distinct for the maternal homolog. The non-naturally occurringnucleotides corresponding to the paternal homolog share a feature “p”which is distinct for the paternal homolog.

As shown in FIG. 11, moieties are added to the non-naturally occurringnucleotides, such as detectable labels. The maternal non-naturallyoccurring nucleotides can have the same moiety, such as the same label.The maternal non-naturally occurring nucleotides can have the samemoiety, such as the same label. But, the labels may be different todifferentially label the maternal and paternal homologs. According toone aspect, moieties other than detectable labels can be added. Certainmoieties can include antibodies, ligands, enzymes etc. for any desiredfunction.

As shown in FIG. 12, non-naturally occurring nucleotides are designed toaccept additional moieties, labels, nucleotides etc. In one aspect,non-naturally occurring nucleotides including a detectable label may beadded in series in order to augment a signal. According to one aspect, acertain non-naturally occurring nucleotide is design to accept onlynon-naturally occurring nucleotides of the same type.

FIG. 13 is a schematic embodiment of FIG. 12 showing non-naturallyoccurring nucleotides extending from a non-naturally occurringnucleotides corresponding to SNPs on the maternal homolog.

FIG. 14 is a schematic embodiment of FIG. 12 showing a singlenon-naturally occurring nucleotide type extending from two differentnon-naturally occurring nucleotides corresponding to SNPs on thematernal homolog. In this manner, a labeled non-naturally occurringnucleotide can be a dual purpose label.

FIG. 15 is a schematic embodiment of FIG. 12 showing a singlenon-naturally occurring nucleotide type extending from two differentnon-naturally occurring nucleotides corresponding to SNPs on thematernal homolog and showing a single non-naturally occurring nucleotidetype extending from two different non-naturally occurring nucleotidescorresponding to SNPs on the paternal homolog. In this manner, a labelednon-naturally occurring nucleotide can be a dual purpose label.

FIG. 16 is a schematic showing differentiation of a maternal homologfrom a paternal homolog using labeling of sequences which differ betweenthe maternal homolog and the paternal homolog. According to this aspect,homologs can differ by the presence of one of a sequence that is notpresent on the other homolog. One example of such a change would be adeletion from one homolog and/or an insertion on the other. If such achange is large enough to be detected by FISH or another marker, then itcan be used to distinguish one homolog from another by using differentdetectable labels, (such as red and green labels). According to thisaspect, homologs can differ by many sequence differences.

FIG. 17 is a schematic showing ligation across SNPs on the maternalhomolog and the paternal homolog with the result being a labelednucleotide being hybridized to a SNP and in a manner to differentiatethe maternal homolog from the paternal homolog. According to thisaspect, two oligonucleotides are used to flank and cover a SNP where oneof the oligos includes the complement to the SNP base. Oligonucleotidesare used for the various SNPs on the maternal and paternal homologs. Thelabeled nucleotide for the maternal homolog is different from thelabeled nucleotide for the parental homolog and therefore, both homologscan be differentially labeled after ligation.

As shown in FIG. 18, non-naturally-occurring nucleotides can be used inthe ligation procedure.

As shown in FIG. 19, labeled probes can be directly hybridized to SNPsand imaged to different the maternal homolog from the paternal homolog.

As shown in FIG. 20, the labeled probes of FIG. 19 can be labeledtoe-hold probes to differentiate the maternal homolog from the paternalhomolog.

As shown in figure FIG. 21 is a schematic showing amplification of anucleotide complementary to a SNP using a padlock probe including thenucleotide and rolling circle amplification.

FIG. 22 is a schematic showing amplification of a nucleotidecomplementary to a SNP using a padlock probe and ligation to include thenucleotide complementary to the SNP into a template for rolling circleamplification.

FIG. 23 is a schematic showing use of a padlock probe to hybridize acomplementary non-naturally occurring nucleotide to a SNP where thepadlock probe hybridizes flanking the SNP. The probe is ligated acrossthe SNP with the inclusion of the complementary non-naturally occurringnucleotide. The non-naturally occurring nucleotide can then be imaged orotherwise detected using the methods described herein and in a manner todistinguish the maternal homolog from the paternal homolog.

The contents of all references, patents and published patentapplications cited throughout this application are hereby incorporatedby reference in their entirety for all purposes.

EQUIVALENTS

Other embodiments will be evident to those of skill in the art. Itshould be understood that the foregoing description is provided forclarity only and is merely exemplary. The spirit and scope of thepresent invention are not limited to the above example, but areencompassed by the claims. All publications, patents and patentapplications cited above are incorporated by reference herein in theirentirety for all purposes to the same extent as if each individualpublication or patent application were specifically indicated to be soincorporated by reference.

What is claimed is:
 1. A fluorescence in situ hybridization method ofdistinguishing a first gene in a maternal chromosome from a second genein a paternal chromosome by single nucleotide polymorphisms whichdistinguish the first gene from the second gene and wherein the firstgene and the second gene are homologs comprising identifying a firstnucleotide type that is a single nucleotide polymorphism within thefirst gene, hybridizing a first primer type directly upstream of thefirst nucleotide type, extending the first primer type across the firstnucleotide type in the presence of a first polymerase, first extensionnucleotides and a first labeled extension nucleotide complementary tothe first nucleotide type, wherein the first labeled extensionnucleotide hybridizes to the first nucleotide type, identifying a secondnucleotide type that is a single nucleotide polymorphism within thesecond gene and which is different from the first nucleotide type,hybridizing a second primer type directly upstream of the secondnucleotide type, extending the second primer type across the secondnucleotide type in the presence of a second polymerase, second extensionnucleotides and a second labeled extension nucleotide complementary tothe second nucleotide type wherein the second labeled extensionnucleotide hybridizes to the second nucleotide type, wherein the firstgene is differentially labeled from the second gene.
 2. The method ofclaim 1 further including identifying a third nucleotide type that is asingle nucleotide polymorphism within the first gene and which isdifferent from the first nucleotide type and the second nucleotide type,hybridizing a third primer type directly upstream of the thirdnucleotide type, extending the third primer type across the thirdnucleotide type in the presence of a third polymerase, third extensionnucleotides and a third labeled extension nucleotide complementary tothe third nucleotide type, wherein the third labeled extensionnucleotide hybridizes to the third nucleotide type, and wherein thefirst gene is differentially labeled from the second gene.
 3. The methodof claim 1 further including identifying a third nucleotide type that isa single nucleotide polymorphism within the first gene and which isdifferent from the first nucleotide type and the second nucleotide type,hybridizing a third primer type directly upstream of the thirdnucleotide type, extending the third primer type across the thirdnucleotide type in the presence of a third polymerase, third extensionnucleotides and a third labeled extension nucleotide complementary tothe third nucleotide type, wherein the third labeled extensionnucleotide hybridizes to the third nucleotide type, and wherein thefirst gene is differentially labeled from the second gene, wherein aspecific first nucleotide within the first gene is excluded from beinglabeled if its corresponding nucleotide on the second gene is of thesame nucleotide type as the third nucleotide type on the first gene. 4.The method of claim 1 further including identifying a third nucleotidetype that is a single nucleotide polymorphism within the first gene andwhich is different from the first nucleotide type and the secondnucleotide type, hybridizing a third primer type directly upstream ofthe third nucleotide type, extending the third primer type across thethird nucleotide type in the presence of a third polymerase, thirdextension nucleotides and a third labeled extension nucleotidecomplementary to the third nucleotide type, wherein the third labeledextension nucleotide hybridizes to the third nucleotide type,identifying a fourth nucleotide type that is a single nucleotidepolymorphism within the second gene and which is different from thefirst nucleotide type, the second nucleotide type, and the thirdnucleotide type, hybridizing a fourth primer type directly upstream ofthe fourth nucleotide type, extending the fourth primer type across thefourth nucleotide type in the presence of a fourth polymerase, fourthextension nucleotides and a fourth labeled extension nucleotidecomplementary to the fourth nucleotide type wherein the fourth labeledextension nucleotide hybridizes to the fourth nucleotide type, whereinthe first gene is differentially labeled from the second gene.
 5. Themethod of claim 1 further including identifying a third nucleotide typethat is a single nucleotide polymorphism within the first gene and whichis different from the first nucleotide type and the second nucleotidetype, hybridizing a third primer type directly upstream of the thirdnucleotide type, extending the third primer type across the thirdnucleotide type in the presence of a third polymerase, third extensionnucleotides and a third labeled extension nucleotide complementary tothe third nucleotide type, wherein the third labeled extensionnucleotide hybridizes to the third nucleotide type, identifying a fourthnucleotide type that is a single nucleotide polymorphism within thesecond gene and which is different from the first nucleotide type, thesecond nucleotide type, and the third nucleotide type, hybridizing afourth primer type directly upstream of the fourth nucleotide type,extending the fourth primer type across the fourth nucleotide type inthe presence of a fourth polymerase, fourth extension nucleotides and afourth labeled extension nucleotide complementary to the fourthnucleotide type wherein the fourth labeled extension nucleotidehybridizes to the fourth nucleotide type, wherein the first gene isdifferentially labeled from the second gene, wherein a specific firstnucleotide within the first gene is excluded from being labeled if itscorresponding nucleotide on the second gene is of the same nucleotidetype as the third nucleotide type on the first gene, and wherein aspecific second nucleotide within the second gene is excluded from beinglabeled if its corresponding nucleotide on the first gene is of the samenucleotide type as the fourth nucleotide type on the second gene.
 6. Themethod of claim 1 wherein the first labeled extension nucleotide or thesecond labeled extension nucleotide is a modified nucleotide.
 7. Themethod of claim 1 wherein the first labeled extension nucleotide is afirst modified nucleotide and the second labeled extension nucleotide isa second modified nucleotide.
 8. The method of claim 1 wherein the firstlabeled extension nucleotide is a first modified nucleotide and thesecond labeled extension nucleotide is a second modified nucleotide,wherein the first modified nucleotide is attachable only by firstmodified nucleotides of the same type and wherein the second modifiednucleotide is attachable only by second modified nucleotides of the sametype.
 9. The method of claim 4 wherein the first labeled extensionnucleotide is a first modified nucleotide, the second labeled extensionnucleotide is a second modified nucleotide, the third labeled extensionnucleotide is a third modified nucleotide and the fourth labeledextension nucleotide is a fourth modified nucleotide, wherein the firstmodified nucleotide and the third modified nucleotide are attachable bythe same modified nucleotide.
 10. The method of claim 4 wherein thefirst labeled extension nucleotide is a first modified nucleotide, thesecond labeled extension nucleotide is a second modified nucleotide, thethird labeled extension nucleotide is a third modified nucleotide andthe fourth labeled extension nucleotide is a fourth modified nucleotide,wherein the second modified nucleotide and the fourth modifiednucleotide are attachable by the same modified nucleotide.
 11. Themethod of claim 4 wherein the first labeled extension nucleotide is afirst modified nucleotide, the second labeled extension nucleotide is asecond modified nucleotide, the third labeled extension nucleotide is athird modified nucleotide and the fourth labeled extension nucleotide isa fourth modified nucleotide, wherein the first modified nucleotide andthe third modified nucleotide are attachable by the same first attachingmodified nucleotide, wherein the second modified nucleotide and thefourth modified nucleotide are attachable by the same second attachingmodified nucleotide, and wherein the first attaching modified nucleotideis different from the second attaching modified nucleotide.
 12. Themethod of claim 1 wherein the first labeled extension nucleotide or thesecond labeled extension nucleotide is a modified nucleotide to which adetectable label can be added or which can be further modified todistinguish the first gene from the second gene.
 13. The method of claim2 wherein the third labeled extension nucleotide is a modifiednucleotide.
 14. The method of claim 2 wherein the third labeledextension nucleotide is a modified nucleotide to which a detectablelabel can be added or which can be further modified to distinguish thefirst gene from the second gene.
 15. The method of claim 4 wherein thefirst labeled extension nucleotide, the second labeled extensionnucleotide, the third labeled extension nucleotide or the fourth labeledextension nucleotide is a modified nucleotide.
 16. The method of claim 4wherein the first labeled extension nucleotide, the second labeledextension nucleotide, the third labeled extension nucleotide or thefourth labeled extension nucleotide is a modified nucleotide to which adetectable label can be added or which can be further modified todistinguish the first gene from the second gene.
 17. The method of claim4 wherein the modified nucleotide of the first labeled extensionnucleotide and the third labeled extension nucleotide is different fromthe modified nucleotide of the second labeled extension nucleotide andthe fourth labeled extension nucleotide such that the modifiednucleotide hybridized to the first gene is different from the modifiednucleotide hybridized to the second gene.
 18. A fluorescence in situhybridization method of distinguishing a first gene in a maternalchromosome from a second gene in a paternal chromosome by sequencespresent in one gene and not the other and wherein the first gene and thesecond gene are homologs comprising identifying sequence differencesbetween the first gene and the second gene, hybridizing first labeledprobes to sequences present in the first gene but not present in thesecond gene, hybridizing second labeled probes to sequences present inthe second gene but not in the first gene, wherein the first label isdifferent from the second label and the first gene is differentiallylabeled compared to the second gene.
 19. A fluorescence in situhybridization method of distinguishing a first gene in a maternalchromosome from a second gene in a paternal chromosome by singlenucleotide polymorphisms which distinguish the first gene from thesecond gene and wherein the first gene and the second gene are homologscomprising identifying a first nucleotide type that is a singlenucleotide polymorphism within a first sequence within the first gene,hybridizing a first labeled complementary sequence to the first sequenceand with a first labeled nucleotide of the first labeled complementarysequence hybridizing to the first nucleotide type, hybridizing a firstcomplementary sequence to the first sequence and adjacent to the firstlabeled complementary sequence, ligating the first labeled complementarysequence to the first sequence, identifying a second nucleotide typethat is a single nucleotide polymorphism within a second sequence withinthe second gene, hybridizing a second labeled complementary sequence tothe second sequence and with a second labeled nucleotide of the secondlabeled complementary sequence hybridizing to the second nucleotidetype, hybridizing a second complementary sequence to the second sequenceand adjacent to the second labeled complementary sequence, ligating thesecond labeled complementary sequence to the second sequence, whereinthe first gene is differentially labeled from the second gene.
 20. Themethod of claim 19 wherein the first labeled nucleotide is a firstmodified nucleotide and the second labeled nucleotide is a secondmodified nucleotide.
 21. The method of claim 19 wherein the firstlabeled nucleotide is a first modified nucleotide and the second labelednucleotide is a second modified nucleotide, wherein the first modifiednucleotide is only extendable by first modified nucleotides of the sametype and wherein the second modified nucleotide is only extendable bysecond modified nucleotides of the same type.
 22. The method of claim 19wherein the first labeled nucleotide or the second labeled nucleotide isa modified nucleotide to which a detectable label can be added or whichcan be further modified to distinguish the first gene from the secondgene.
 23. A fluorescence in situ hybridization method of distinguishinga first gene in a maternal chromosome from a second gene in a paternalchromosome by single nucleotide polymorphisms which distinguish thefirst gene from the second gene and wherein the first gene and thesecond gene are homologs comprising identifying a first nucleotide typethat is a single nucleotide polymorphism within a first sequence withinthe first gene, hybridizing a first labeled complementary sequence tothe first sequence and with a first labeled nucleotide of the firstlabeled complementary sequence hybridizing to the first nucleotide type,hybridizing a first complementary sequence to the first sequence andadjacent to the first labeled complementary sequence, ligating the firstlabeled complementary sequence to the first sequence, identifying asecond nucleotide type that is a single nucleotide polymorphism within asecond sequence within the second gene, hybridizing a second labeledcomplementary sequence to the second sequence and with a second labelednucleotide of the second labeled complementary sequence hybridizing tothe second nucleotide type, hybridizing a second complementary sequenceto the second sequence and adjacent to the second labeled complementarysequence, ligating the second labeled complementary sequence to thesecond sequence, identifying a third nucleotide type that is a singlenucleotide polymorphism within a third sequence within the first geneand which is different from the first nucleotide type and the secondnucleotide type, hybridizing a third labeled complementary sequence tothe third sequence and with a third labeled nucleotide of the thirdlabeled complementary sequence hybridizing to the third nucleotide type,hybridizing a third complementary sequence to the third sequence andadjacent to the third labeled complementary sequence, ligating the thirdlabeled complementary sequence to the third sequence, identifying afourth nucleotide type that is a single nucleotide polymorphism within afourth sequence within the first gene and which is different from thefirst nucleotide type, the second nucleotide type and the thirdnucleotide type, hybridizing a fourth labeled complementary sequence tothe fourth sequence and with a fourth labeled nucleotide of the fourthlabeled complementary sequence hybridizing to the fourth nucleotidetype, hybridizing a fourth complementary sequence to the fourth sequenceand adjacent to the fourth labeled complementary sequence, ligating thefourth labeled complementary sequence to the fourth sequence, andwherein the first gene is differentially labeled from the second gene.24. The method of claim 23 wherein the first labeled nucleotide is afirst modified nucleotide, the second labeled nucleotide is a secondmodified nucleotide, the third labeled nucleotide is a third modifiednucleotide and the fourth labeled nucleotide is a fourth modifiednucleotide, wherein the first modified nucleotide and the third modifiednucleotide are extendable by the same modified nucleotide.
 25. Themethod of claim 23 wherein the first labeled nucleotide is a firstmodified nucleotide, the second labeled nucleotide is a second modifiednucleotide, the third labeled nucleotide is a third modified nucleotideand the fourth labeled nucleotide is a fourth modified nucleotide,wherein the second modified nucleotide and the fourth modifiednucleotide are extendable by the same modified nucleotide.
 26. Themethod of claim 23 wherein the first labeled nucleotide is a firstmodified nucleotide, the second labeled nucleotide is a second modifiednucleotide, the third labeled nucleotide is a third modified nucleotideand the fourth labeled nucleotide is a fourth modified nucleotide,wherein the first modified nucleotide and the third modified nucleotideare extendable by the same first extension modified nucleotide, whereinthe second modified nucleotide and the fourth modified nucleotide areextendable by the same second extension modified nucleotide, and whereinthe first extension modified nucleotide is different from the secondextension modified nucleotide.
 27. The method of claim 23 wherein thefirst labeled nucleotide, the second labeled nucleotide, the thirdlabeled nucleotide or the fourth labeled nucleotide is a modifiednucleotide.
 28. The method of claim 23 wherein the first labelednucleotide, the second labeled nucleotide, the third labeled nucleotideor the fourth labeled nucleotide is a modified nucleotide to which adetectable label can be added or which can be further modified todistinguish the first gene from the second gene.
 29. The method of claim27 wherein the modified nucleotide of the first labeled nucleotide andthe third labeled nucleotide is different from the modified nucleotideof the second labeled nucleotide and the fourth labeled nucleotide suchthat the modified nucleotide hybridized to the first gene is differentfrom the modified nucleotide hybridized to the second gene.
 30. Afluorescence in situ hybridization method of distinguishing a first genein a maternal chromosome from a second gene in a paternal chromosome bysingle nucleotide polymorphisms which distinguish the first gene fromthe second gene and wherein the first gene and the second gene arehomologs comprising identifying a first nucleotide type that is a singlenucleotide polymorphism within the first gene, hybridizing a first probeincluding a first labeled nucleotide complementary to the firstnucleotide type, identifying a second nucleotide type that is a singlenucleotide polymorphism within the second gene and which is differentfrom the first nucleotide type, hybridizing a second probe including asecond labeled nucleotide complementary to the second nucleotide type,wherein the first gene is differentially labeled from the second gene.31. The method of claim 30 wherein a first toe-hold probe is hybridizedto the first gene with the first probe and a second toe-hold probe ishybridized to the second gene with the second probe.
 32. A fluorescencein situ hybridization method of distinguishing a first gene in amaternal chromosome from a second gene in a paternal chromosome bysingle nucleotide polymorphisms which distinguish the first gene fromthe second gene and wherein the first gene and the second gene arehomologs comprising identifying a first nucleotide type that is a singlenucleotide polymorphism within the first gene, hybridizing a first probeincluding a first labeled nucleotide complementary to the firstnucleotide type, identifying a second nucleotide type that is a singlenucleotide polymorphism within the second gene and which is differentfrom the first nucleotide type, hybridizing a second probe including asecond labeled nucleotide complementary to the second nucleotide type,identifying a third nucleotide type that is a single nucleotidepolymorphism within the first gene and which is different from the firstnucleotide type and the second nucleotide type, hybridizing a thirdprobe including a third labeled nucleotide complementary to the thirdnucleotide type, identifying a fourth nucleotide type that is a singlenucleotide polymorphism within the second gene and which is differentfrom the first nucleotide type, the second nucleotide type, and thethird nucleotide type, hybridizing a fourth probe including a fourthlabeled nucleotide complementary to the fourth nucleotide type, whereinthe first gene is differentially labeled from the second gene.
 33. Themethod of claim 32 wherein a first toe-hold probe is hybridized to thefirst gene with the first probe, a second toe-hold probe is hybridizedto the second gene with the second probe, a third toe-hold probe ishybridized to the first gene with the third probe, and a fourth toe-holdprobe is hybridized to the second gene with the fourth probe.
 34. Afluorescence in situ hybridization method of distinguishing a first genein a maternal chromosome from a second gene in a paternal chromosome bysingle nucleotide polymorphisms which distinguish the first gene fromthe second gene and wherein the first gene and the second gene arehomologs comprising identifying a first nucleotide type that is a singlenucleotide polymorphism within the first gene, hybridizing a firstamplifiable probe to a first sequence including the first nucleotidetype and including a first labeled nucleotide complementary to the firstnucleotide type, amplifying the first amplifiable probe to produce oneor more amplicons including the first labeled nucleotide, identifying asecond nucleotide type that is a single nucleotide polymorphism withinthe second gene and which is different from the first nucleotide type,hybridizing a second amplifiable probe to a second sequence includingthe second nucleotide type and including a second labeled nucleotidecomplementary to the second nucleotide type, amplifying the secondamplifiable probe to produce one or more amplicons including the secondlabeled nucleotide, wherein the amplicons including the first nucleotidetype are differentially labeled from the amplicons including the secondnucleotide type.
 35. The method of claim 34 wherein the firstamplifiable probe is a padlock probe and the padlock probe is amplifiedusing rolling circle amplification.
 36. The method of claim 34 whereinthe second amplifiable probe is a padlock probe and the padlock probe isamplified using rolling circle amplification.
 37. A fluorescence in situhybridization method of distinguishing a first gene in a maternalchromosome from a second gene in a paternal chromosome by singlenucleotide polymorphisms which distinguish the first gene from thesecond gene and wherein the first gene and the second gene are homologscomprising identifying a first nucleotide type that is a singlenucleotide polymorphism within the first gene, hybridizing a first endand a second end of first amplifiable probe to first sequences flankingthe first nucleotide type, extending and ligating nucleotides from thefirst end of the first amplifiable probe to the second end of the firstamplifiable probe and across the first nucleotide type and including afirst labeled nucleotide complementary to the first nucleotide type,amplifying the first amplifiable probe to produce one or more ampliconsincluding the first labeled nucleotide, identifying a second nucleotidetype that is a single nucleotide polymorphism within the second gene andwhich is different from the first nucleotide type, hybridizing a firstend and a second end of second amplifiable probe to second sequencesflanking the second nucleotide type, extending and ligating nucleotidesfrom the first end of the second amplifiable probe to the second end ofthe second amplifiable probe and across the second nucleotide type andincluding a second labeled nucleotide complementary to the firstnucleotide type, amplifying the second amplifiable probe to produce oneor more amplicons including the second labeled nucleotide, wherein theamplicons including the first nucleotide type are differentially labeledfrom the amplicons including the second nucleotide type.
 38. The methodof claim 37 wherein the first amplifiable probe is a padlock probe andthe padlock probe is amplified using rolling circle amplification. 39.The method of claim 37 wherein the first amplifiable probe is a padlockprobe and the padlock probe is amplified using rolling circleamplification.
 40. A fluorescence in situ hybridization method ofdistinguishing a first gene in a maternal chromosome from a second genein a paternal chromosome by single nucleotide polymorphisms whichdistinguish the first gene from the second gene and wherein the firstgene and the second gene are homologs comprising identifying a firstnucleotide type that is a single nucleotide polymorphism within thefirst gene, hybridizing a first end and a second end of a first probe tofirst sequences flanking the first nucleotide type, extending andligating nucleotides from the first end of the first probe to the secondend of the first probe and across the first nucleotide type andincluding a first non-naturally occurring nucleotide complementary tothe first nucleotide type, identifying a second nucleotide type that isa single nucleotide polymorphism within the second gene and which isdifferent from the first nucleotide type, hybridizing a first end and asecond end of a second probe to second sequences flanking the secondnucleotide type, extending and ligating nucleotides from the first endof the second probe to the second end of the second probe and across thesecond nucleotide type and including a second non-naturally occurringnucleotide complementary to the first nucleotide type, wherein the firstnon-naturally occurring nucleotide is different from the secondnon-naturally occurring nucleotide.
 41. A fluorescence in situhybridization method of distinguishing a first gene in a maternalchromosome from a second gene in a paternal chromosome by sequencedifferences which distinguish the first gene from the second gene andwherein the first gene and the second gene are homologs comprisingidentifying a first nucleotide type that indicates a sequence differencewithin the first gene, hybridizing a first primer type directly upstreamof the first nucleotide type, extending the first primer type across thefirst nucleotide type in the presence of a first polymerase, firstextension nucleotides and a first labeled extension nucleotidecomplementary to the first nucleotide type, wherein the first labeledextension nucleotide hybridizes to the first nucleotide type,identifying a second nucleotide type that indicates a sequencedifference within the second gene and which is different from the firstnucleotide type, hybridizing a second primer type directly upstream ofthe second nucleotide type, extending the second primer type across thesecond nucleotide type in the presence of a second polymerase, secondextension nucleotides and a second labeled extension nucleotidecomplementary to the second nucleotide type wherein the second labeledextension nucleotide hybridizes to the second nucleotide type, whereinthe first gene is differentially labeled from the second gene.
 42. Themethod of claim 41 further including identifying a third nucleotide typethat indicates a sequence difference within the first gene and which isdifferent from the first nucleotide type and the second nucleotide type,hybridizing a third primer type directly upstream of the thirdnucleotide type, extending the third primer type across the thirdnucleotide type in the presence of a third polymerase, third extensionnucleotides and a third labeled extension nucleotide complementary tothe third nucleotide type, wherein the third labeled extensionnucleotide hybridizes to the third nucleotide type, and wherein thefirst gene is differentially labeled from the second gene.
 43. Themethod of claim 41 further including identifying a third nucleotide typethat indicates a sequence difference within the first gene and which isdifferent from the first nucleotide type and the second nucleotide type,hybridizing a third primer type directly upstream of the thirdnucleotide type, extending the third primer type across the thirdnucleotide type in the presence of a third polymerase, third extensionnucleotides and a third labeled extension nucleotide complementary tothe third nucleotide type, wherein the third labeled extensionnucleotide hybridizes to the third nucleotide type, and wherein thefirst gene is differentially labeled from the second gene, wherein aspecific first nucleotide within the first gene is excluded from beinglabeled if its corresponding nucleotide on the second gene is of thesame nucleotide type as the third nucleotide type on the first gene. 44.The method of claim 41 further including identifying a third nucleotidetype that indicates a sequence difference within the first gene andwhich is different from the first nucleotide type and the secondnucleotide type, hybridizing a third primer type directly upstream ofthe third nucleotide type, extending the third primer type across thethird nucleotide type in the presence of a third polymerase, thirdextension nucleotides and a third labeled extension nucleotidecomplementary to the third nucleotide type, wherein the third labeledextension nucleotide hybridizes to the third nucleotide type,identifying a fourth nucleotide type that indicates a sequencedifference within the second gene and which is different from the firstnucleotide type, the second nucleotide type, and the third nucleotidetype, hybridizing a fourth primer type directly upstream of the fourthnucleotide type, extending the fourth primer type across the fourthnucleotide type in the presence of a fourth polymerase, fourth extensionnucleotides and a fourth labeled extension nucleotide complementary tothe fourth nucleotide type wherein the fourth labeled extensionnucleotide hybridizes to the fourth nucleotide type, wherein the firstgene is differentially labeled from the second gene.
 45. A fluorescencein situ hybridization method of distinguishing a first gene in amaternal chromosome from a second gene in a paternal chromosome bysequence differences which distinguish the first gene from the secondgene and wherein the first gene and the second gene are homologscomprising identifying a first nucleotide type that indicates a sequencedifference within the first gene, hybridizing a first primer typedirectly upstream of the first nucleotide type, extending the firstprimer type across the first nucleotide type in the presence of a firstpolymerase, first extension nucleotides and a first labeled extensionnucleotide complementary to a nucleotide in the sequence difference,wherein the first labeled extension nucleotide hybridizes to thenucleotide in the sequence difference, identifying a second nucleotidetype that indicates a sequence difference within the second gene andwhich is different from the first nucleotide type, hybridizing a secondprimer type directly upstream of the second nucleotide type, extendingthe second primer type across the second nucleotide type in the presenceof a second polymerase, second extension nucleotides and a secondlabeled extension nucleotide complementary to a nucleotide in thesequence difference wherein the second labeled extension nucleotidehybridizes to the nucleotide in the sequence difference, wherein thefirst gene is differentially labeled from the second gene.
 46. Themethod of claim 45 further including identifying a third nucleotide typethat indicates a sequence difference within the first gene and which isdifferent from the first nucleotide type and the second nucleotide type,hybridizing a third primer type directly upstream of the thirdnucleotide type, extending the third primer type across the thirdnucleotide type in the presence of a third polymerase, third extensionnucleotides and a third labeled extension nucleotide complementary to anucleotide in the sequence difference, wherein the third labeledextension nucleotide hybridizes to the nucleotide in the sequencedifference, and wherein the first gene is differentially labeled fromthe second gene.
 47. The method of claim 45 further includingidentifying a third nucleotide type that indicates a sequence differencewithin the first gene and which is different from the first nucleotidetype and the second nucleotide type, hybridizing a third primer typedirectly upstream of the third nucleotide type, extending the thirdprimer type across the third nucleotide type in the presence of a thirdpolymerase, third extension nucleotides and a third labeled extensionnucleotide complementary to a nucleotide in the sequence difference,wherein the third labeled extension nucleotide hybridizes to thenucleotide in the sequence difference, and wherein the first gene isdifferentially labeled from the second gene, wherein a specific firstnucleotide within the first gene is excluded from being labeled if itscorresponding nucleotide on the second gene is of the same nucleotidetype as the third nucleotide type on the first gene.
 48. The method ofclaim 45 further including identifying a third nucleotide type thatindicates a sequence difference within the first gene and which isdifferent from the first nucleotide type and the second nucleotide type,hybridizing a third primer type directly upstream of the thirdnucleotide type, extending the third primer type across the thirdnucleotide type in the presence of a third polymerase, third extensionnucleotides and a third labeled extension nucleotide complementary to anucleotide in the sequence difference, wherein the third labeledextension nucleotide hybridizes to the nucleotide in the sequencedifference, identifying a fourth nucleotide type that indicates asequence difference within the second gene and which is different fromthe first nucleotide type, the second nucleotide type, and the thirdnucleotide type, hybridizing a fourth primer type directly upstream ofthe fourth nucleotide type, extending the fourth primer type across thefourth nucleotide type in the presence of a fourth polymerase, fourthextension nucleotides and a fourth labeled extension nucleotidecomplementary a nucleotide in the sequence difference wherein the fourthlabeled extension nucleotide hybridizes to the nucleotide in thesequence difference, wherein the first gene is differentially labeledfrom the second gene.
 49. A fluorescence in situ hybridization method ofdistinguishing a first gene in a maternal chromosome from a second genein a paternal chromosome by sequence differences which distinguish thefirst gene from the second gene and wherein the first gene and thesecond gene are homologs comprising identifying a first sequencedifference within the first gene, hybridizing a first primer typedirectly upstream of the first sequence difference, extending the firstprimer type across the first sequence difference in the presence of afirst polymerase, first extension nucleotides and a first labeledextension nucleotide complementary to a nucleotide in the first sequencedifference, wherein the first labeled extension nucleotide hybridizes tothe nucleotide in the first sequence difference, identifying a secondsequence difference within the second gene and which is different fromthe first sequence difference, hybridizing a second primer type directlyupstream of the second sequence difference, extending the second primertype across the second sequence difference in the presence of a secondpolymerase, second extension nucleotides and a second labeled extensionnucleotide complementary to a nucleotide in the second sequencedifference wherein the second labeled extension nucleotide hybridizes tothe nucleotide in the second sequence difference, wherein the first geneis differentially labeled from the second gene.
 50. The method of claim49 further including identifying a third sequence difference within thefirst gene and which is different from the first sequence difference andthe second sequence difference, hybridizing a third primer type directlyupstream of the third sequence difference, extending the third primertype across the third sequence difference in the presence of a thirdpolymerase, third extension nucleotides and a third labeled extensionnucleotide complementary to a nucleotide in the third sequencedifference, wherein the third labeled extension nucleotide hybridizes tothe nucleotide in the third sequence difference, and wherein the firstgene is differentially labeled from the second gene.
 51. The method ofclaim 49 further including identifying a third sequence differencewithin the first gene and which is different from the first sequencedifference and the second sequence difference, hybridizing a thirdprimer type directly upstream of the third sequence difference,extending the third primer type across the third sequence difference inthe presence of a third polymerase, third extension nucleotides and athird labeled extension nucleotide complementary to a nucleotide in thethird sequence difference, wherein the third labeled extensionnucleotide hybridizes to the nucleotide in the third sequencedifference, and wherein the first gene is differentially labeled fromthe second gene.
 52. The method of claim 49 further includingidentifying a third nucleotide type that indicates a third sequencedifference within the first gene and which is different from the firstsequence difference and the second sequence difference, hybridizing athird primer type directly upstream of the third sequence difference,extending the third primer type across the third sequence difference inthe presence of a third polymerase, third extension nucleotides and athird labeled extension nucleotide complementary to a nucleotide in thethird sequence difference, wherein the third labeled extensionnucleotide hybridizes to the nucleotide in the third sequencedifference, identifying a fourth nucleotide type that indicates a fourthsequence difference within the second gene and which is different fromthe first sequence difference, the second sequence difference, and thethird sequence difference, hybridizing a fourth primer type directlyupstream of the fourth sequence difference, extending the fourth primertype across the fourth sequence difference in the presence of a fourthpolymerase, fourth extension nucleotides and a fourth labeled extensionnucleotide complementary a nucleotide in the fourth sequence differencewherein the fourth labeled extension nucleotide hybridizes to thenucleotide in the fourth sequence difference, wherein the first gene isdifferentially labeled from the second gene.